Scalable primate pluripotent stem cell aggregate suspension culture and differentiation thereof

ABSTRACT

The present invention relates to methods for production of undifferentiated or differentiated embryonic stem cell aggregate suspension cultures from undifferentiated or differentiated embryonic stem cell single cell suspensions and methods of differentiation thereof.

RELATED APPLICATIONS

This application is a Continuation-in-Part under 35 U.S.C. §120 of U.S.patent application Ser. No. 13/220,590, filed Aug. 29, 2011, which is aContinuation of U.S. patent application Ser. No. 12/264,760, filed Nov.4, 2008, now U.S. Pat. No. 8,008,075, issued Aug. 30, 2011, thedisclosures of which are incorporated herein by reference in theentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Part of the work performed during development of this invention utilizedU.S. Government funds from National Institutes of Health Grant No. 5R24RR021313-05. The U.S. Government has certain rights in thisinvention.

FIELD OF THE INVENTION

The present invention relates to suspension cell aggregate compositionsthat are essentially serum and feeder-free and methods fordifferentiating the cell aggregate suspensions.

BACKGROUND OF THE INVENTION

To date, there is no efficient system providing for a large-scalemanufacturing process (“scale-up”) for mammalian pluripotent cells suchas human embryonic stem cells (hESC) as described herein. To maintainhESC in an undifferentiated state in vitro, the hESC are maintained onmouse embryonic fibroblast (MEF) feeders and passaged by manualmechanical dissociation (e.g., micro-dissection) and transferringindividual colony pieces. These methods are sufficient for researchstudies that do not require large-scale production of undifferentiatedhESC or differentiated hESC, gene targeting, drug discovery, in vitrotoxicology, future clinical applications require improved methods forthe stable large-scale expansion of hESC, including enzymatic passaging.

Enzymatic expansion of hESC can be performed but these methods havetechnical disadvantages because hESC depend on cell-cell interactions aswell as para- and autocrine signals for survival. Hence, hESC preferthis cellular microenvironment as compared to existing as single cells.Also, there are reports that enzymatic dissociation of hESC may lead toabnormal karyotypes and result in genetic and epigenetic changes. Thus,providing a highly supportive culture environment while at the same timeallowing for robust large-scale expansion (i.e., a manufacturingprocess) of undifferentiated hES or differentiated hESC withoutcompromising the pluripotency, multipotency or genetic stability overextended culture periods is essential.

Human pluripotent cells offer unique opportunities for investigatingearly stages of human development as well as for therapeuticintervention in several disease states, such as diabetes mellitus andParkinson's disease. For example, the use of insulin-producing β-cellsderived from hESC would offer a vast improvement over current celltherapy procedures that utilize cells from donor pancreases. Currentlycell therapy treatments for diabetes mellitus, which utilize cells fromdonor pancreases, are limited by the scarcity of high quality isletcells needed for transplant. Cell therapy for a single Type I diabeticpatient requires a transplant of approximately 8×10⁸ pancreatic isletcells (Shapiro et al. 2000, N Engl J Med 343:230-238; Shapiro et al.2001a, Best Pract Res Clin Endocrinol Metab 15:241-264; Shapiro et al.2001, British Medical Journal 322:861). As such, at least two healthydonor organs are required to obtain sufficient islet cells for asuccessful transplant.

hESC thus represent a powerful model system for the investigation ofmechanisms underlying pluripotent cell biology and differentiationwithin the early embryo, as well as providing opportunities for geneticmanipulation of mammals and resultant commercial, medical andagricultural applications. Furthermore, appropriate proliferation anddifferentiation of hESC can potentially be used to generate an unlimitedsource of cells suited to transplantation for treatment of diseases thatresult from cell damage or dysfunction. Other pluripotent cells and celllines including early primitive ectoderm-like (EPL) cells as describedin International Patent Application WO 99/53021, in vivo or in vitroderived ICM/epiblast, in vivo or in vitro derived primitive ectoderm,primordial germ cells (EG cells), teratocarcinoma cells (EC cells), andpluripotent cells derived by dedifferentiation or by nuclear transferwill share some or all of these properties and applications.International Patent Application WO 97/32033 and U.S. Pat. No. 5,453,357describe pluripotent cells including cells from species other thanrodents. Human ES cells have been described in International PatentApplication WO 00/27995, and in U.S. Pat. No. 6,200,806, and human EGcells have been described in International Patent Application WO98/43679.

The biochemical mechanisms regulating ES cell pluripotency anddifferentiation are very poorly understood. However, the limitedempirical data available (and much anecdotal evidence) suggests that thecontinued maintenance of pluripotent ES cells under in vitro cultureconditions is dependent upon the presence of cytokines and growthfactors present in the extracellular milieu.

While human ESCs offer a source of starting material from which todevelop substantial quantities of high quality differentiated cells forhuman cell therapies, these cells must be obtained and/or cultured inconditions that are compatible with the expected regulatory guidelinesgoverning clinical safety and efficacy. Such guidelines likely willrequire the use of media with all components sourced with cGMP. Thedevelopment of such chemically defined/GMP standard conditions isnecessary to facilitate the use of hESCs and cells derived from hESCsfor therapeutic purposes in humans.

In addition, the eventual application of hESC based cell replacementtherapies will require the development of methods that enable largescale culture and differentiation conditions that are compliant withregulatory guidelines. While several groups have reported simplifiedgrowth conditions for hESCs, there are substantial limitations withthese studies. To date, however, the successful isolation, long-termclonal maintenance, genetic manipulation and germ line transmission ofpluripotent cells has generally been difficult.

Most of the cell culture conditions for stem cells still contain serumreplacer (KSR) in the media (Xu et al. 2005 Stem Cells, 23:315-323; Xuet al. 2005 Nature Methods, 2:185-189; Beattie et al. 2005 Stem Cells,23:489-495; Amit et al. 2004 Biol. Reprod., 70:837-845; James et al.2005 Development, 132:1279-1282). KSR contains a crude fraction ofbovine serum albumin (BSA) rather than a highly purified source. Othershave only performed short-term studies, and therefore it is not clear iftheir conditions would enable the maintenance of pluripotency overextended periods (Sato et al. 2004, Nature Med. 10:55-63; U.S. PatentPublication Nos. 2006/0030042 and 2005/0233446). Others have shownlong-term maintenance of pluripotency in a chemically defined media withFGF2, activin A, and insulin, but the cells were grown on plates thatwere coated with human serum, which was “washed off” before plating ofcells (Vallier et al. 2005 J Cell Sci., 118(Pt 19):4495-509). While FGF2has been a component of all these media, it is not clear if it providesa primary or secondary self-renewal signal (Bendall et al. 2007 Nature448:1015-1027); particularly as in some formulations it is necessary touse it at a high concentration (up to 100 ng/mL, Xu et al. 2005 NatureMethods, 2:185-189).

Furthermore, all of these groups have either included insulin in theirmedia at μg/mL levels, or have insulin present due to the use of KSR.Insulin is typically considered to function in glucose metabolism and“cell survival” signaling via binding to the insulin receptor. At levelsabove physiological concentrations, however, insulin can also bind tothe IGF1 receptor with a lower efficiency and confer classical growthfactor activity through the PI3 Kinase/AKT pathway. Thepresence/requirement for such high levels of insulin (μg/mL levels) inKSR or these other media conditions suggests that the major activity iselicited via binding to the IGF1 receptor, which is expressed by hESCs(Sperger et al. 2003 PNAS, 100(23):13350-13355). Others have noted theexpression of a full complement of IGF1R and intracellular signalingpathway members in hESCs, which is likely to signify the functionalactivity of this pathway (Miura et al. 2004 Aging Cell, 3:333-343).Insulin or IGF1 may elicit a major signal required for the self-renewalof hESCs, as is suggested by the fact that all conditions developed thusfar for the culture of hESC contain either insulin, insulin provided byKSR, or IGF1 provided by serum. In support of this concept, it has beenshown that if PI3 Kinase is inhibited in hESC cultures, the cellsdifferentiate (D'Amour et al. 2005, Nat. Biotechnol 23:1534-41; McLeanet al. 2007 Stem Cells 25:29-38).

A recent publication outlines a humanized, defined media for hESCs(Ludwig et al. Nature Biotechnology, published online Jan. 1, 2006,doi:10.1038/nbt1177). This recent formulation, however, includes severalfactors that are suggested to influence the proliferation of hESCs,including FGF2, TGFβ, LiCl, γ-aminobutyric acid and pipecolic acid. Itis noted that this recently defined cell culture medium also containsinsulin.

A self-renewal signaling paradigm for hESC based on a combination ofinsulin/IGF1, heregulin, Activin A signaling was previously reported byApplicant. See Wang et al. 2007 Blood 110:4111-4119. In this context wehave found that an exogenous FGF2 signal is redundant and not required(Schulz & Robins 2009, supra) Schulz & Robins 2009, (In: Lakshmipathy etal. eds., Emerging Technology Platforms for Stem Cells. John Wiley &Sons., Hoboken, N.J., pp. 251-274);) Heregulin is a member of the EGFgrowth factor family. There are at least 14 members, including, but notlimited to, EGF, TGFβ, heparin binding-EGF (hb-EGF), neuregulin-β (alsonamed heregulin-β (HRG-β), glial growth factor and others), HRG-α,amphiregulin, betacellulin, and epiregulin. All these growth factorscontain an EGF domain and are typically first expressed as transmembraneproteins that are processed by metalloproteinase (specifically, ADAM)proteins to generate soluble ectodomain growth factors. EGF familymembers interact with either homo- or hetero-dimers of the ErbB1, 2, 3and 4 cell surface receptors with different affinities (Jones et al.FEBS Lett, 1999, 447:227-231). EGF, TGFα and hbEGF bind ErbB1/1 (EGFR)homodimers and ErbB1/2 heterodimers at high affinity (1-100 nM range),whereas HRG-β binds ErbB3 and ErbB4 at very high affinity (<1 nM range).Activated ErbB receptors signal through the PI3 Kinase/AKT pathway andalso the MAPK pathway. ErbB2 and ErbB3 are amongst the most highlyexpressed growth factor receptors in hESCs (Sperger et al. 2003, PNAS,100:13350-13355) and HRG-β has been shown previously to support theexpansion of mouse primordial germ cells (Toyoda-Ohno et al. 1999, Dev.Biol., 215:399-406). Furthermore, over expression and subsequentinappropriate activation of ErbB2 is associated with tumorigenesis (Neveet al. 2001 Ann. Oncol, 12(Suppl 1):S9-13; Zhou & Hung, 2003 Semin.Oncol. 30(5 Suppl 16):38-48; Yarden, 2001, Oncology, 61 Suppl 2:1-13).Human ErbB2 (Chromosome 17q), and ErbB3 (Chromosome 12q) are present onchromosomes that have been observed to accumulate as trisomies in somehESCs (Draper et al. 2004 Nat. Biotechnol. 22:53-4; Cowan et al. 2004 NEngl. J. Med. 350(13):1353-6; Brimble et al. 2004 Stem Cells Dev.,13:585-97; Maitra et al. 2005 Nat. Genet. 37:1099-103; Mitalipova et al.2005 Nat. Biotechnol. 23: 19-20; Draper et al. 2004 Stem Cells Dev.,13:325-36; Ludwig et al. Nature Biotech, published online Jan. 1, 2006,doi:10.1038/nbt1177).

ErbB2 and ErbB3 (Brown et al. 2004 Biol. Reprod., 71:2003-11;Salas-Vidal & Lomeli, 2004 Dev Biol. 265:75-89) are expressed in themouse blastocyst, although not specifically restricted to the inner cellmass (ICM), and ErbB1, EGF and TGFβ are expressed in the humanblastocyst (Chia et al. 1995 Development, 1221(2):299-307). HB-EGF hasproliferative effects in human IVF blastocyst culture (Martin et al.1998 Hum. Reprod. 13:1645-52; Sargent et al. 1998 Hum. Reprod. 13(Suppl4):239-48), and modest additional effects on mouse ES cells grown in 15%serum (Heo et al. 2006 Am. J. Phy. Cell Physiol. 290:C123-33, Epub 2005Aug. 17. Pre- and early post-implantation development does not appear tobe affected in ErbB2−/−, ErbB3−/−, Neuregulin1−/− (Britsch et al. 1998Genes Dev., 12:1825-36), ADAM17−/− (Peschon et al. 1998 Science, 282:1281-1284) and ADAM19−/− (Horiuchi 2005 Dev. Biol. 283:459-71) nullembryos. Therefore, the importance of signaling through the ErbBreceptor family in hESCs is, up to now, unclear.

Neuregulin-1 (NRG1) is a large gene that exhibits multiple splicing andprotein processing variants. This generates a large number of proteinisoforms, which are referred to herein collectively as neuregulin.Neuregulin is predominantly expressed as a cell surface transmembraneprotein. The extracellular region contains an immunoglobulin-likedomain, a carbohydrate modified region and the EGF domain. NRG1expression isoforms have been reviewed previously (Falls 2003 Exp. CellRes. 284:14-30). The cell membrane metalloproteases ADAM17 and ADAM19have been shown to process the transmembrane form(s) of neuregulin-1 tosoluble neuregulin/heregulin. HRG-α and -β are the cleaved ectodomainsof neuregulin, containing the EGF and other domains. As the EGF domainis responsible for binding and activation of the ErbB receptors, arecombinant molecule containing only this domain can exhibit essentiallyall of the soluble growth factor effects of this protein (Jones et al.1999 FEBS Lett. 447:227-31). Also, there are processed transmembraneisoforms of neuregulin that are thought to trigger juxtacrine signalingin adjacent cells via interaction of the EGF domain with ErbB receptors.

Still, an important development in the progression of hESC researchtoward maintaining pluripotency in culture will be the elucidation ofmedia and cell culture conditions that are compatible with the expectedregulatory guidelines governing clinical safety and efficacy. While thebest outcome would be the availability of chemically defined media forhESC, components that are not chemically defined would be acceptable ifthey were produced to GMP standard. There is a need, therefore, toidentify methods and compositions for the culture and stabilization of apopulation of pluripotent stem cells that are able to be used fortherapeutic purposes, wherein the culture compositions are definedand/or produced to GMP standard.

The production of committed progenitor or differentiated cell types thatcan function following transplantation is a central promise of thepotential of hESC-based therapeutic research. Using a step-wiseprotocol, in particular a 4-stage step-wise protocol substantiallysimilar to that described herein and previously in Applicant's patentand non-patent publications, also referred to herein, primatepluripotent stem cells (pPSC) e.g., hESC or iPSC, are differentiablecells that can be directed to differentiate to a mixed population ofpancreatic type cells by the end of stage 4. The mixture of cellscontains at least cells commonly referred to as “pancreaticprogenitors”, or “pancreatic endoderm”, or “pancreatic epithelium” bothalso referred to as “PE”, or “PDX1-positive pancreatic endoderm”, or“pancreatic endoderm cells” or “PEC” or equivalents thereof.

The cellular composition of PEC has been fully characterized asdescribed in Applicant's prior patent and non-patent applications,including but not limited to Kroon et al. 2008 Nature Biotechnology26:443-52, and U.S. Pat. Nos. 7,534,608; 7,695,965; and 7,993,920,entitled METHODS FOR MAKING INSULIN IN VIVO, and 8,278,106, entitledENCAPSULATION OF PANCREATIC CELLS DERIVED FROM HUMAN PLURIPOTENT STEMCELLS, which are herein incorporated by reference in their entireties.Using flow cytometry, quantification of more than 20 samples from morethan 10 different development lots of PEC showed the following types ofcells. About 50% (ranges from 33-60%) of the cell mixture consisted ofcells that express NKX6-1 but not Chromogranin (CHGA). About 44% (range33-62%) poly-hormonal endocrine cells express CHGA. CHGA positive cellshave been shown to develop and mature to glucagon expressing cellsfollowing in vivo transplantation or implantation. About 7% (range1.3-13%) express PDX1 while at the same time do not express CHGA orNKX6-1 (PDX1 only population). A very small group of cells, about 1%(range 0.27-6.9%) in the mixture or population express none of the abovemarkers: neither PDX1, nor NKX6-1, nor CHGA (or triple negative cells).Hence, PEC or equivalents thereof refers to this population or mixtureof cells. PEC composition or population is also described in more detailin Example 27 and Table 12. Kroon et al. 2008, supra, Schulz et al.2012, supra, which disclosures are all incorporated herein by referencein their entireties.

Implanted PEC, encapsulated or un-encapsulated, gives rise tofunctioning islet-like structures in vivo through a mechanism thatappears to primarily involve the de novo commitment of pancreaticprogenitors to the endocrine lineages followed by further maturation toglucose-responsive β-cells. Such grafts are therefore capable of sensingblood glucose, responding with metered release of processed humaninsulin, and protecting against streptozotocin (STZ)-inducedhyperglycemia in mice. See Kroon et al. 2008, supra.

While other candidate pancreatic lineages have been derived from hESC,none have demonstrated substantial post-engraftment function in vivo, asdefined by both long-term glucose-responsive human c-peptide secretionand protection against STZ-induced hyperglycemia. Without demonstratedfunction in animal models, it is difficult to gauge the scalability, orclinical potential, of these alternate protocols. See Cai J. et al. 2009J Mol Cell Biol 2:50-60; Johannesson et al. 2009 PLoS One 4:e4794;Mfopou et al. 2010 Gastroenterology 138: 2233-2245; Ungrin et al. 2011Biotechnol Bioeng. December 2. doi:10.1002/bit.24375; Clark et al. 2007Biochem Biophys Res Commun 356:587-593; Jiang et al. 2007 Cell Res 17:333-344; and Shim et al. 2007 Diabetologia 50:1228-1238, which areincorporated herein by reference in their entireties.

The invention described herein follows on Applicant's previousdemonstration that feeder-free conditions using defined media cansupport single cell passaging and bulk culture of hESC. See Schulz &Robins 2009, supra; and U.S. Pat. No. 8,278,106, entitled ENCAPSULATIONOF PANCREATIC CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS, which areherein incorporated by reference in their entireties. Critical for theprogression of hESC-based technology to clinical trials is ademonstration of comparable scalability. Improvements that enhanceexpansion efficiencies will also save time and produce cost savings, aswell as minimize the potential for population drift over time spent inculture. See Maitra et al. 2005 Nat Genet. 37:1099-1103, which isincorporated herein by reference in its entirety. Importantly, scalingusing roller bottles as described herein, for example, along withcryopreservation of hESC, provides a defined and consistent material forproduct manufacture for near and long term research and developmentstrategies.

SUMMARY OF THE INVENTION

The invention relates to compositions comprising a basal salt nutrientsolution and an ErbB3 ligand, with the compositions being essentiallyfree of serum.

The invention also relates to compositions comprising a basal saltnutrient solution and a means for stimulating ErbB2-directed tyrosinekinase activity in differentiable cells.

The invention relates to methods of culturing differentiable cells, withthe methods comprising plating the differentiable cells on a cellculture surface, providing a basal salt nutrient solution to thedifferentiable cells and providing a ligand that specifically bindsErbB3.

The invention relates to methods of culturing differentiable cells, withthe methods comprising plating the differentiable cells on a cellculture surface and providing a basal salt nutrient solution to thedifferentiable cells and a means for stimulating ErbB2-directed tyrosinekinase activity in the differentiable cells.

The invention also relates to methods of culturing differentiable cells,with the methods comprising providing a digest solution to a layer ofdifferentiable cells that are contained in a culture chamber prior todigestion, where the digestion breaks apart the layer of cells intosingle cells. After digestion, the single cells are placed into a newtissue culture chamber with a differentiable cell culture solution,wherein the differentiable cell culture solution comprises a basal saltnutrient solution and an ErbB3 ligand. Once cultured, the singledifferentiable cells are placed in conditions that permit growth anddivision of the single cells.

The invention relates to methods for generating a hES cell aggregate insuspension from a pluripotent hES adherent culture, by culturing a hEScell in an adherent growth culture condition which allows for expansionin an undifferentiated state; disassociating the adherent hES cellculture into a single cell suspension culture; contacting the singlecell suspension culture with a first differentiating culture conditionwhich allows for formation of hES-derived cell aggregates in suspensionby agitating the single cell suspension culture until such a period oftime when the single cell suspension culture forms a hES-derived cellaggregate in suspension, and thereby generating a hES-derived cellaggregate in suspension. In preferred embodiments, agitation of thesingle cell suspension culture is performed by rotation at about 80 rpmto 160 rpm

The invention also relates to methods for generating a hES-derived cellaggregate in suspension from a hES-derived single cell suspension, byculturing a hES cell in an adherent growth culture condition whichallows for expansion in an undifferentiated state; contacting theundifferentiated hES cell with a first differentiating culturingcondition suitable for differentiating the hES cell and resulting in anadherent hES-derived cell; disassociating the adherent hES-derived cellinto a single cell suspension culture; contacting the single cellsuspension culture with a second differentiating culture condition whichallows for formation of hES-derived cell aggregates in suspension byagitating the single cell suspension culture until such a period of timewhen the single cell suspension culture forms a hES-derived cellaggregate in suspension, and thereby generating a hES-derived cellaggregate in suspension. In preferred embodiments, agitation of thesingle cell suspension culture is performed by rotation at about 80 rpmto 160 rpm.

The invention relates to a roller bottle containing primate pluripotentstem cell (pPSC) aggregates in suspension. In certain aspects of theinvention, the pPSC aggregates are cells selected from the groupconsisting of human embryonic stem cells (hESC), induced pluripotentstem cells (iPSC) and/or other human pluripotent stem cells. In oneembodiment, the roller bottle is not vented, but can be vented dependingon the incubator or oven capabilities.

The invention also relates to methods for generating a roller bottlecontaining pPSC aggregates by contacting pPSCs with a pluripotent stemcell culture condition, and agitating the culture until pPSC aggregatesform, thereby generating pPSC aggregates in the roller bottle. Incertain embodiments, agitation of the pPSC culture is performed byrotation at about 3 rpm, about 4 rpm, about 5 rpm, about 6 rpm, about 7rpm, about 8 rpm, about 9 rpm, about 10 rpm, about 11 rpm, about 12 rpm,about 13 rpm, about 14 rpm, about 15 rpm, about 16 rpm, about 17 rpm,about 18 rpm, about 19 rpm, about 20 rpm, about 21 rpm, about 22 rpm,about 23 rpm, about 24 rpm, about 25 rpm, about 26 rpm, about 27 rpm,about 28 rpm, about 29 rpm and about 30 rpm. Typically, agitation of thepPSC culture is performed by rotation at about 5 rpm, about 6 rpm, about7 rpm, about 8 rpm, about 9 rpm, about 10 rpm, about 11 rpm, and about12 rpm.

Another aspect of the invention relates to methods for differentiatingpPSC aggregates in a roller bottle by contacting differentiable orundifferentiated pPSC aggregates with a culturing condition thatdifferentiates the pPSCs, and agitating the pPSC aggregate culture untilformation of pPSC-derived aggregates, thereby generating pPSC-derivedaggregates in suspension in a roller bottle. In certain embodiments,agitation of the pPSC-derived aggregates suspension culture is performedby rotation at about 3 rpm, about 4 rpm, about 5 rpm, about 6 rpm, about7 rpm, about 8 rpm, about 9 rpm, about 10 rpm, about 11 rpm, about 12rpm, about 13 rpm, about 14 rpm, about 15 rpm, about 16 rpm, about 17rpm, about 18 rpm, about 19 rpm, about 20 rpm, about 21 rpm, about 22rpm, about 23 rpm, about 24 rpm, about 25 rpm, about 26 rpm, about 27rpm, about 28 rpm, about 29 rpm and about 30 rpm. Typically, agitationof the pPSC culture is performed by rotation at about 5 rpm, about 6rpm, about 7 rpm, about 8 rpm, about 9 rpm, about 10 rpm, about 11 rpm,and about 12 rpm.

Still another embodiment of the invention relates to methods where fluidflow within a rolling bottle type of vessel involves rolling movementthat does not require rotation or rolling the bottle. In one embodiment,the rolling type movement is substantially re-created but without theuse of a rolling vessel. In another embodiment, a primate pluripotentstem cell culture has imparted fluid movement, for example, by pumpingor flowing a fluid in a smooth, orderly manner with little or noturbulence. In such embodiments, any sub-current generally moves inparallel with any other nearby sub-current(s). This type of movement isalso characterized as laminar flow (commonly used to move viscousfluids, especially those moving at low velocities) or streamline flow (asteady movement of fluid movement). In a yet another embodiment, thefluid movement involves one or more baffles, which distribute the fluidflow within a chamber to create a continuous, uniform suspension ofcells. In a still further embodiment, the fluid movement involves one ora combination of deflector plates, distribution channels, and/or flowchannels. In each embodiment, there is included at least one or moreseals on the culture vessel to ensure an aseptic environment inside thevessel during cell aggregation, growth and differentiation.

The invention also relates to methods for enriching or varying thecomposition of the resulting cell culture and/or population of anhES-derived cell aggregate suspension by optimizing the cell density ofthe pluripotent cell cultures or varying the concentration of variousgrowth factors, for example, FGF10, EGF, KGF, noggin and retinoic acid,apoptotic inhibitors, Rho-kinase inhibitors and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts real time RT-PCR expression analysis of ADAM19,Neuregulin1, and ErbB1-3 in BG01v grown in defined conditions (8 ng/mLFGF2, 100 ng/mL LR-IGF1, 1 ng/mL Activin A). GAPDH and OCT4 controlreactions are indicated.

FIG. 2 depicts the inhibition of proliferation of BG01v cells usingAG879. BG01v cells were plated in 6-well trays and exposed to DMSO (A),50 nM-20 μM AG1478 (B), or 100 mM-20 μM AG879 (C) 24 hours afterplating. After 5 days in culture, the cultures were fixed and stainedfor alkaline phosphatase activity. AG1478 did not appear to affectproliferation at these concentrations (20 μM shown in B), but AG879substantially slowed cell growth at 5 μM (C).

FIG. 3 depicts the morphology of BG01v cells cultured in DC-HAIF, whichis defined culture media containing 10 ng/mL HRG-β, 10 ng/mL Activin A,200 ng/mL LR-IGF1 and 8 ng/mL FGF2 (A and B), and in defined culturemedia (DC) containing 10 ng/mL HRG-β, 10 ng/mL Activin A, and 200 ng/mLLR-IGF1 (C and D).

FIG. 4 depicts the expression of ADAM19, Neuregulin1, and ErbB1-4 byRT-PCR in mouse ES cells (A) and MEFs (B).

FIG. 5 depicts the inhibition of ErbB1 and ErbB2 signaling in mouse EScells. 2×10⁵ Mouse R1 ES cells were plated on 1:1000 MATRIGEL™ in 10%FBS, 10% KSR with 1000 U/mL mouse LIF (ESGRO). The following day, DMSO(carrier control), 1-50 μM AG1478, or 1-50 μM AG879 was added with freshmedium. The cultures were fixed on day 8, and stained for alkalinephosphatase activity. DMSO (A) and 1-50 μM AG1478 (B and C) did notovertly inhibit proliferation. AG879 substantially inhibited cell growthat 50 μM (compare D and F) and may have slowed proliferation at 20 μM(E).

FIG. 6 depicts the inhibition of proliferation of BG02 cells grown inconditioned media (CM). (A) 50 μM AG825 inhibited proliferation of BG02hESCs growing in CM. (B) AG825 inhibits ErbB2 Y1248 phosphorylation inhESCs. (C) Colony counting of serial passaging of CyT49 hESCs indifferent combinations of growth factors. (D) Cell counting analysis ofthe role of IGF1 and HRG in hESC proliferation using BG02 cells (left).(E) OCT4/DAPI immunostaining of a duplicate repeated experimentdemonstrated that IGF1 and HRG significantly increased the proportion ofOCT4⁺ cells compared to ActA/FGF2 conditions. (F) RTK blotting analysisof BG01 DC-HAIF hESCs starved of growth factors overnight; starved, thenpulsed with DC-HAIF for 15 minutes; or steady-state cultures are shown(left). The mean and range of normalized relative intensity is plotted(right).

FIG. 7 depicts mouse ES cells grown in defined conditions with differentgrowth factor combinations. (A) shows the scoring of AP⁺ colonies after2×10⁵ cells were grown in different growth factor combinations for 8days. (B-G) show 4× magnification images of AP⁺ colonies grown indifferent growth factor combinations.

FIG. 8 depicts the characterization of human ES cells that aremaintained in DC-HAIF medium. (A) Analysis of teratomas from BG02DC-HAIF p25 cells demonstrated pluripotent differentiation potential toectoderm, mesoderm and endoderm. (B) Immunostaining of BG02 cellscultured in 15% FCS/5% KSR that have differentiated. (C) Venn diagram ofthe distribution of transcripts detected using high density IlluminaSentrix Human-6 Expression Beadchips containing 47,296 transcript probesin BG02 cells maintained in CM (64 passages) or DC-HAIF (10 or 32passages in defined media). (D) Scatterplot analysis demonstrating thatthe transcriptional profile of BG02 DC-HAIF p32 cells is highly similarto that of BG02 cells maintained in CM (top), and was not substantiallyaltered in early and late passage cultures in DC-HAIF (bottom). (E)Hierarchical clustering dendrogram of relative gene expression indifferent populations generated using the Beadstudio software.

FIG. 9 depicts the morphology of cells cultured on humanizedextracellular matrices (ECMs) in the presence of DC-HAIF medium. (A)CyT49 cells (diluted 1:200) growing on growth factor-reduced MATRIGEL™(diluted 1:200). CyT49 cells could also grow on tissue culture dishescoated with (B) whole human serum, (C) human fibronectin, and (D)VITROGROT™.

FIG. 10 depicts the single-cell passaging of human ES cells. (A-D)Staged imaging of BG02 cells after passaging with ACCUTASE™ and platingabout 5×10⁵ cells in a 60 mm culture dish. (A) 1.5 hours after initialplating, showing viable cells adhering to the dish. (B) At 20 hourspost-plating, the large majority of cells have aggregated to form smallcolonies. These colonies expand by proliferation by day 4, post-plating(C), and over the course of 5-6 days to form an epithelial-likemonolayer covering the entire dish (D). (E) Normal male karyotypedemonstrated in a BG02 culture passaged 19 times with ACCUTASE™ inDC-HAIF.

FIG. 11 depicts cell morphology after single cell passaging of human EScells using (A) ACCUTASE™, (B) 0.25% Trypsin/EDTA, (C) TrypLE, or (D)Versene.

FIG. 12 depicts the large-scale growth of human ES cells cultured inDC-HAIF. (A) Flow cytometric analysis of BG02 cells after expansion to>10¹⁰ cells. >85% of cells expressed OCT4, CD9, SSEA-4, TRA-1-81. (B)RT-PCR analysis of expression of markers of pluripotency OCT4, NANOG,REX1, SOX2, UTF1, CRIPTO, FOXD3, TERT and DPPA5. Markers ofdifferentiated lineages, α-fetoprotein (AFP), MSX1 and HAND1 were notdetected. (C) Fluorescence in situ hybridization (FISH) using humanchromosome-specific repeats demonstrated maintenance of normal copynumbers for hChr 12, 17, X and Y.

FIG. 13 depicts the morphology (A) and normal karyotype (B) of hESC BG02cells grown in defined media comprising HRG-β and IGF1, but in theabsence of FGF2 for 7 passages, or >2 months.

FIG. 14 depicts a scatter plot analysis of transcripts from hESCs (BG02)that are maintained in DC-HAIF (32 passages) or DC-HAI (10 passages). Alarge proportion of the expressed transcripts were detected in bothsamples, and transcription was not substantially altered by culturinghESCs in the absence of exogenous FGF2. Correlation coefficients (R²)were generated using all detected transcripts with an expression levelof >0 (all dots), or with transcripts exhibiting a detection confidencelevel of >0.99 (R² select, dots indicated by dashed oval). Angled linesdelineate the mean and limits of a 2-fold difference.

FIG. 15 depicts a hierarchical clustering dendrogram of relative geneexpression in different populations of early and late passage BG02 cellsmaintained in DC-HAIF. Cells clustered tightly (˜0.0075) and retained aclose similarity to BG02 and BG03 cells maintained in conditioned medium(CM) (˜0.037). BG02 cells maintained in DC-HAI also clustered tightlywith the other hESC populations examined. By way of explanation in FIG.15, CM is Conditioned Medium; DC is defined culture medium, DC-HAIF asdefined above; ap is ACCUTASE™ single cell passaging; DC-HAI isidentical to DC-HAIF as defined herein, except without FGF2.

FIG. 16 depicts the morphology and alkaline phosphatase staining of BG02cells cultured in DC-HAIF in 96-well and 384-well plates. (A) Phasecontrast imaging and (B) alkaline phosphatase staining of BG02 cells(10⁴ cells/well) growing in one well of a 96-well plate. (C) Phasecontrast imaging and (D) alkaline phosphatase staining of BG02 cells(10³ cells/well) growing in one well of a 384-well plate.

FIG. 17 depicts dark field images of BG02 grown in DC-HAIF in suspensionculture. Day 2 and day 6 cultures are shown. The images were capturedusing 4× magnification

FIG. 18 depicts the growth rates in adherent and suspension cultures inDC-HAIF. 1×10⁶ BG02 cells were plated into parallel wells in adherentand suspension culture and cell counts were performed on days 1-6.

FIG. 19 depicts qPCR analysis of suspension and adherent hESCs. BG02cells growing in suspension (S. hESCs) and adherent (hESCs) cultureexhibited comparable levels of OCT4, and lacked SOX17 expression.Adherent cells differentiated to definitive endoderm (DE), andsuspension hESCs differentiated to definitive endoderm in suspension (S.DE d3), both exhibited the expected marked down regulation of OCT4 andup regulation of SOX17 expression

FIG. 20 depicts the enhancement of hESC aggregation in the presence ofY27632 in suspension culture. 2×10⁶ BG02 cells were seeded in 3 mLDC-HAIF or DC-HAIF+Y27632, in 6-well trays, in an incubator on arotating platform at 100 rpm. Images of aggregates were captured on days1 and 3.

FIG. 21 depicts RT-PCR analysis of suspension aggregates in the presenceof Y27632. RT-PCR was performed on the expanded cultures to assessexpression of markers of pluripotency. Expression of OCT4, NANOG, REX1,SOX2, UTF1, CRIPTO, FOXD3, TERT AND DPPA5 was detected, whereas markersof differentiated lineages AFP, MSX1 and HAND1 were not detected.

FIGS. 22 A-N are bar charts showing the expression patterns of markergenes OCT4 (panel A), BRACH (panel B), SOX17 (panel C), FOXA2 orHNF3beta (panel D), HNF 1beta (panel E), PDX1 (panel F) NKX6.1 (panelG), NKX2.2 (panel H), INS (panel I), GCG (panel J), SST (panel K), SOX7(panel L), ZIC1 (panel M), AFP (panel N), HNF4A (panel O) and PTF1A(panel P), which is not an exhaustive list but markers which can be usedto identify pluripotent human embryonic stem (hES) cells (stage0, d0),definitive endoderm cells (stage1; d2), PDX1-negative foregut endodermcells (stage2; d5), PDX1-postiive endoderm cells (stage3, d8),pancreatic endoderm cells (stage4; d11), pancreatic endocrine precursorsand/or hormone secreting cells (stage5; d15).

FIG. 23 is a graph showing the range of the diameters of the cellaggregates in suspension (microns) in relationship to the total volume(mL) of media in the culture.

FIGS. 24 A-D are bar charts showing the expression patterns of markergenes PDX1 (panel A) NKX6.1 (panel B), NGN3 (panel C) and NKX2.2 (panelD) in hES-derived cells in relationship to the cell density of the hEScell cultures from which they were derived.

FIG. 25 is a chart showing cell aggregate diameters of pluripotent cellsat day zero (d0) and differentiating cell aggregates at days 2, 5, 8 and12 (d2, d5, d8 and d12, respectively). Cell aggregate sizes weremeasured and plotted showing the minimum, maximum, 2nd and 3rd quartile,and median. Each day shows the plot for cell aggregates formed from1×10⁶ cells/mL (left) and 2×10⁶ cells/mL (right).

FIGS. 26A-D are bar charts showing the expression patterns of thevarious indicated marker genes in rolling bottle vessel format. The leftsample of each chart represents day zero (d0) cell aggregates formed in6-well trays (pluripotent cell marker control). The samples marked bybars represent (left to right): undifferentiated aggregates at day 0,and differentiating aggregates at days 2, 5, 8 and 12. Black bar,rolling bottles at 1×10⁶ cells/mL; black dashed bar, rolling bottles at2×10⁶ cells/mL; grey bar, 6-well tray.

FIGS. 27A-D are bar charts showing the expression patterns of thevarious indicated marker genes in larger rolling bottle vessel formatsas described in Table 11 and 12 in Example 27. The left samplerepresents a 6-well tray hESC aggregation and differentiation (control;FIG. 27A); Differentiation at days 0, 2, 5, 8 and 12 in vented (V) ornot-vented (NV) 490 cm² roller bottles (about 1.2 L capacity), areshown. Day 2 samples were not collected for the last 490V sample (farright column) due to loss of culture. The same d0 control was used foreach roller bottle differentiation (asterisk).

DETAILED DESCRIPTION OF THE INVENTION

In contrast to previously known methods of tissue engineering which arebased on seeding individual cells into polymer scaffolds, matricesand/or gels, the methods described herein use cell aggregate suspensionsformed from pluripotent hES single cell suspensions or hES-derived(differentiated) single cell suspensions as the building blocks oftissue formation. Cell aggregates are often comprised of hundreds tothousands of individual cells, connected through junctional adhesionsand extracellular matrix that collectively contribute to the finaldifferentiated product. In this regard, cell aggregates can be definedas a type of tissue that provides a number of performance advantagesrelative to more traditional engineered tissues.

In one embodiment of the invention, methods are provided for producinghES cell aggregate suspensions from a single cell suspension ofpluripotent stem cell cultures or hES-derived cell cultures. Thepluripotent stem cells can be initially cultured on fibroblast feeders,or they can be feeder-free. Methods of isolating hESC and culturing suchon human feeder cells was described in U.S. Pat. No. 7,432,104 entitledMETHODS FOR THE CULTURE OF HUMAN EMBRYONIC STEM CELLS ON HUMAN FEEDERCELLS, which is herein incorporated in its entirety by reference.Pluripotent ES cell aggregate suspension cultures made directly orinitiated from hESCs cultured on feeders avoid the need for making hESCmonolayers, for example, as in adherent cultures. These methods aredescribed in detail in Examples 17 and 18.

Other embodiments of the invention provide for methods of producing cellaggregate suspensions directly into a differentiation media, e.g., adifferentiating media containing an agent, preferably a TGFβ familymember, which is capable of activating a TGFβ family of receptor. Suchagents include but are not limited Activin A, Activin B, GDF-8, GDF-11,and Nodal. Methods of producing cell aggregate suspension in adifferentiation media is distinguished from other methods, alsodescribed herein, which provide for production of cell aggregatesuspension cultures in a pluripotent stem cell media, e.g., StemPro.

Still other embodiments of the invention provide for methods ofproducing cell aggregate suspensions formed from differentiated hES cellcultures (also referred to as “hES-derived cell cultures” or“hES-derived cell(s)”), e.g., cells from stages 1, 2, 3, 4 and 5 asdescribed in D'Amour et al. 2005, supra and D'Amour et al. 2006, NatureBiotech 26 2006: 1392-1401). Hence, methods for making the cellaggregates described herein are not limited to any one pluripotent ormultipotent stage of a hES or hES-derived cell, rather the manner of useand need for cell type optimization will dictate which methods arepreferred. These methods are described in detail in Examples 19-22.

In another embodiment of the invention, methods are provided forcontrolling the resulting cell composition, e.g., controlling thepercentage of pancreatic endoderm cells, pancreatic endocrine cellsand/or PDX1-endoderm cells, by varying the concentration of differentgrowth factors. These methods are described in detail in Example 21.

Unless otherwise noted, the terms used herein are to be understoodaccording to conventional usage by those of ordinary skill in therelevant art. In addition to the definitions of terms provided below,definitions of common terms in molecular biology may also be found inRieger et al. 1991 Glossary of genetics: classical and molecular, 5thEd., Berlin: Springer-Verlag; and in Current Protocols in MolecularBiology, F. M. Ausubel et al. Eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(1998 Supplement). It is to be understood that as used in thespecification and in the claims, “a” or “an” can mean one or more,depending upon the context in which it is used. Thus, for example,reference to “a cell” can mean that at least one cell can be utilized.

Also, for the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities of ingredients,percentages or proportions of materials, reaction conditions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about”.Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

“About” as used herein means that a number referred to as “about”comprises the recited number plus or minus 1-10% of that recited number.For example, “about” 100 cells can mean 95-105 cells or as few as 99-101cells depending on the situation. Whenever it appears herein, anumerical range such as “1 to 20” refers to each integer in the givenrange; e.g., “1 to 20 cells” means 1 cell, 2 cells, 3 cells, etc., up toand including 20 cells. Where about modifies a range expressed innon-integers, it means the recited number plus or minus 1-10% to thesame degree of significant figures expressed. For example, about 1.50 to2.50 mM can mean as little as 1.35 M or as much as 2.75M or any amountin between in increments of 0.01.

The present invention provides methods for production of hES-derivedcell aggregates from hES-derived single cell suspensions. Becausevarious mechanical and non-physiological factors effect movement andaggregation of cells in culture, the fluid mechanical micro-environmentthat correlates with optimal cell aggregate viability and performance,as well as to provide a normalizing variable that can be used forscale-up, it was necessary to characterize the movement of cells growingor differentiating in various culture vessels, dishes, Erlenmeyerflasks, bioreactors, bottles and the like and the effects, if any, ofvarious media conditions on the cells. Some of these factors include butare not limited to, shear rate and shear stress, cell density andconcentration of various growth factors in any cell medium.

Shear rate and shear stress are mechanical characteristics that definethe fluid shear within a system. Shear rate is defined as the fluidvelocity over a given distance and is expressed as sec⁻¹. Shear rate isproportional to shear stress where shear rate ( )=shear stress(t)/viscosity (μ). Shear stress is defined as the fluid shear forceacting tangentially to the cell surface and is expressed as force perunit area (dyne/cm² or N/m²). Shear stress can be generated by agitatedliquid moving past static cells, agitated cells moving through staticliquid or by cells moving within an agitated, dynamic fluid environment.Fluid viscosity is typically measured in poise where 1 poise=1 dynesec/cm²=100 centipoise (cp). The viscosity of water, one of the leastviscous fluids known, is 0.01 cp. The viscosity of a typical suspensionof eukaryotic cells in media is between 1.0 and 1.1 cp at a temperatureof 25° C. Both density and temperature can affect the viscosity of afluid.

Fluid velocity also dictates whether the flow will be laminar orturbulent. Laminar flow occurs when viscous forces dominate and ischaracterized by smooth, even streamlines at low velocities. Incontrast, high velocity and inertial forces dominate during turbulentflow, which is characterized by the appearance of eddies, vortices andchaotic fluctuations in the flow across space and time. A dimensionlessvalue known as the Reynold's number (Re) is typically used to quantifythe presence of laminar or turbulent flow. The Reynold's number is theratio of inertial to viscous forces and is quantitated as(density*velocity*length scale)/(viscosity). Laminar flow dominates withRe<2300 while turbulent flow dominates when Re>4000. Based on thisrelationship with fluid velocity, the Reynold's number and thus thedegree to which fluid flow is laminar or turbulent is directlyproportional to the shear rate and shear stress experienced by cells insuspension. However, high shear stress conditions can be generated inboth laminar and turbulent fluid environments. Initially, there is atendency for liquid to resist movement, with the fluid closest to asolid surface experiencing attractive forces that generate a boundarylayer or a region of no-flow immediately adjacent to the surface. Thiscreates a gradient in fluid velocity from the surface to the center ofthe fluid flow. The steepness of the velocity gradient is a function ofthe speed at which the liquid is moving and distance from the boundarylayer to the region of highest fluid velocity. As the liquid flow ratethrough or around a container accelerates, the velocity of the flowovercomes the viscosity of the liquid and the smooth, laminar gradientbreaks down producing turbulent flow. Thomas et al. showed that celllysis under turbulent conditions occurs most frequently in regions oflocally high shear stress and high energy dissipation rates. See Thomaset al. (1994) Cytotechnology 15: 329-335. These regions appear randomlybut are often found near the boundary layer where the velocity gradientis highest. These random fluctuations in fluid velocity can generateregions of very high shear stress that ultimately can have a negativeeffect on the scale-up of cell culture-based manufacturing systems.Thus, a need exists for methods that can maintain cell density andviability in a mammalian cell culture manufacturing scale-up system bycontrolling the major sources of shear forces in such systems.

Following methods provided by Henzler (Henzler, 2000, Particle stress inbioreactors, In Advances in Biochemical Engineering/Biotechnology,Scheper, T. Ed. Springer-Verlag, Berlin) and Colomer et al. (Colomer, J.et al. 2005. Experimental analysis of coagulation of particles underlow-shear flow. Water Res. 39:2994), fluid mechanical properties of thebulk fluid in a rotating E-well dish were calculated. The DimensionlessStress is equal to the turbulence constant*(aggregatediameter/Kolmogorov's Microscale)̂turbulence exponent. Shear Stress isequal to the Dimensionless Stress*fluid density*(kinematicviscosity*power input)̂0.5. Shear Rate is equal the ShearStress/kinematic viscosity. For calculation of the power input andKolmogorov's Microscale, the Reynold's number is required at eachrotation rate and is equal to the (rotation rate*flaskdiameter)̂2/viscosity. As both the power input and Kolmogorov'sMicroscale are functions of the Reynold's Number, all shear stress andshear rate calculations vary with rotation rate.

Moreover, shear stress and shear rate are functions of the DimensionlessStress, which depends on the diameter of forming aggregates, thus theshear stress and rate experienced by aggregates is expected to increasewith time in rotation. Example calculations are shown in Example 17 foraggregate diameters between 100-200 μm and rotation speeds between60-140 rpm. These methods were used to provide an estimation of theaverage shear in the bulk fluid over time. However, it is expected thatthe shear stress at the wall of the vessel will be the highest due toboundary effects. To estimate wall shear stress, Ley et al. proposedthat wall shear stress in a 6-well dish is equal to the radius ofgyration*(density*dynamic viscosity*(2*pi*rotation rate)̂3)̂0.5. Usingthis approach, the wall shear stress was calculated for rotation speedsranging from 60 rpm to 140 rpm and is shown in Example 18. Note that,unlike the time-averaged shear stress that is experienced by aggregatesin the bulk fluid, the shear stress occurring at the wall is independentof aggregate diameter.

Culture cell density is also a factor critical to the tissue functionand is difficult to achieve and/or optimize in traditional tissue whichare 2-dimensional (e.g., adherent engineered constructs). The effect ofcell density on differentiation is described in more detail in Example20. Cell aggregates may overcome this limitation by assuming anorganized 3-dimensional (3D) architecture that more accurately reflectsan in vivo cellular density and conformation. As a result, the period oftime for the cells to achieve their intended structure can besignificantly reduced and/or made more consistent and efficient.Moreover, cells in the 3D aggregate format may differentiate andfunction more optimally, as this architecture more closely resemblesnormal physiology than adherent cultures. In addition, the mechanicalhardship involved in the manufacturing process is less damaging to cellaggregates that are free-floating in suspension culture as compared tothe mechanical hardship, for example, in an adherent culture.

Typical manufacturing-scale suspension culture also utilizes continuousperfusion of media as a method for maintaining cell viability whilemaximizing cell density. In this context, media exchange contributesfluid shear to the culture affecting adherent cells and suspendedaggregates differently. Immobile adherent cells are subject to fluidshear stress as the media flows tangentially across the cell surface. Incontrast, suspended aggregates experience significantly less shearstress across the aggregate surface, as aggregates are free to tumble inresponse to applied shear force. It is expected that prolonged shearstress will be detrimental to adherent ES cells and that the suspendedaggregate format is preferred for optimal survival and function. Thusbased on a need for an efficient manufacturing process for production ofpluripotent stem cells and/or multipotent progenitor cells derived frompluripotent stem cells and the above observed mechanics relating toshear rate and shear stress, the present invention provides for thefirst time methods of manufacturing for production of pluripotent stemcells and/or multipotent progenitor cells derived from pluripotent stemcells in suspension format, in particular, cell aggregate suspensionformat.

As used herein, “single cell suspension” or equivalents thereof refersto a hES cell single cell suspension or a hES-derived single cellsuspension by any mechanical or chemical means. Several methods existfor dissociating cell clusters to form single cell suspensions fromprimary tissues, attached cells in culture, and aggregates, e.g.,physical forces (mechanical dissociation such as cell scraper,trituration through a narrow bore pipette, fine needle aspiration,vortex disaggregation and forced filtration through a fine nylon orstainless steel mesh), enzymes (enzymatic dissociation such as trypsin,collagenase, Acutase and the like), or a combination of both. Further,methods and culture media conditions capable of supporting single-celldissociation of hESC is useful for expansion, cell sorting, and definedseeding for multi-well plate assays and enable automatization of cultureprocedures and clonal expansion. Thus, one embodiment of the inventionprovides methods for generating a stable single-cell enzymaticdissociation hES cell or hES-derived cell culture system capable ofsupporting long-term maintenance and efficient expansion ofundifferentiated, pluripotent hES cell or differentiated hESC.

As used herein, “roller bottle” or “rolling bottle” or equivalentsthereof refers to a cylindrical container adapted to rotate about itsaxes. These containers include but are not limited to roller bottlessold through Corning, Fisher Scientific, and other manufacturers, aswell as drums, barrels, and other bottle type containers capable ofbeing rotated on its side wall, for example. Roller bottles describedherein do not have to be cylindrical or have a circular cross-section.They can be non-circular, closed curve, of constant width, for example.In one embodiment, the curve is a Reuleaux triangle or a Reuleauxtriangle with rounded corners as described in U.S. Pat. No. 5,866,419,which is incorporated herein by reference in its entirety. Circularcross-section roller bottles are not the only shape or geometry toprovide smooth rotation because an infinite number of such curves existand are contemplated by the invention. Hover, such curves are notgenerally encountered in industry because most machinery used forrotating bottles requires that the horizontal axis running perpendicularto the curve remain in a fixed location, which it does not fornon-circular rollers because that have axes with a back-and-forthtranslation motion while rolling. This additional motion or rotation, inaddition to the usual circular motion as in other cylindrical rollerbottles can enhance gas exchange as compared to circular cross-sectiontype roller bottles.

A typical cylindrical roller bottle includes a bottom wall, a top walland a cylindrical side wall extending between the bottom and top walls.The top wall includes an opening to provide access to the interior ofthe roller bottle. The internal surfaces of such roller bottles provideactive surfaces for cell interaction and/or attachment. Hence, theOxford Dictionary of Biochemistry provides that roller bottles arecylindrical containers used for the culture of monolayers of adherentcells. Indeed, roller bottles are desirable for growing large amounts ofcells, such as adherent cells, or for producing cell by-products, suchas pharmaceutical substances that are secreted by cells. The cylindricalside wall of roller bottles can be smooth or patterned, wherebypatterning extends substantially from the bottom wall to the top wallfor increasing cell growth surface area and for facilitating the flow ofliquid to all interior surface areas of the bottle when the bottle isrolled about the axis of the side wall.

Independent of the cross-section of the roller bottle (circular ornon-circular) liquid growth medium is introduced into and containedwithin a roller bottle. The rotating movement of the bottle keeps theinternal surfaces wetted with the liquid medium, thereby encouraging thegrowth of cells. Rotating rollers of an appropriate apparatus areemployed to rotate roller bottles of the invention.

Roller bottles are usually constructed of either glass, stainless steelor a clear plastic, such as polystyrene, polyurethane, polyvinylchloride, polycarbonate, polyolefins such as polypropylene, polyethyleneterephthalate with glycol additives, ethylene glycol-1,4, cyclohexanedimethanol terephthalate copolyester and the like. Transparent materialsare preferred, as cell growth can be monitored by placing the bottle onan inverted microscope.

Manual and automated roller bottle systems have been used for over 40years in the pharmaceutical, biochemical, and medical fields forprocesses such as cell growth and infection, heterologous glycoproteinproduction, vaccine preparation, and high density plant cellcultivation. See Tanaka et al. 1983, Biotechnol. Bioeng. 25:2359; Tanaka1987, Process Biochem. August, 106; Hong, et al. 1989, Biotechnol. Prog.5:137; Elliot 1990, Bioprocess Tech. 10:207; Tsao 1992, Annals N.Y.Acad. Sci. 665:127; Pennell & Milstein 1992, J. of Immun. Meth. 146:43;Olivas et al., 1995, Immun. Meth. 182, 73 (1995); Singhvi et al. 1996,Cytotechnology 22:79; and Kunitake et al. 1997, Biotechnology 52:3289,which are incorporated herein by reference in their entireties.Additionally, for industrial scale production of cell culture products(i.e. vaccines), cells are frequently passaged in roller bottles priorto transfer to micro-carrier cultures for a final growth phase even whenunit operation based systems are utilized. See Edy, 1984, Adv. Exp. Med.Biol. 172:169, which is herein incorporated by reference in itsentirety.

To date, widespread use of roller bottles for culturing adherent cellscan be attributed to several factors. The process relies on: (i) ahorizontal cylindrical vessel containing a sufficient volume of media orfluid and axially rotated; because roller bottle scale up is a functionof length, scale-up development or invention is not required, resultingin reduced developmental timelines for industry and faster introductionto market for new products; (ii) roller bottle systems allow forconstant fluid-gas contact, i.e. due to the axial rotation there is atall times at least a thin layer of fluid or media coating the innersurface of the bottle as it rotates; this layer allows for increasedfluid-gas exchange and the as the bottle rotates that gas returns to thecells which are in the pool of media at the bottom of the roller bottle;(iii) maintaining sterile conditions for prolonged times in large scaleculture is possible because contamination of one or more roller bottlesdoes not result in contamination of an entire lot; (iv) precise controlof nutrient and waste-product levels is possible; and (v) directmonitoring of the cells, e.g. identification of certain cell markers toensure efficient differentiation and proper specification of cells afterstages 1-4 for example is relatively simple.

While not wanting to be limited to use of roller bottle or roller typevessels for culturing three-dimensional cell aggregates, it is intendedthat there are other means for making the cell aggregates of theinvention, although not employing the motion created by a roller bottleor a cylindrical type of vessel rotating on a drum, for example. Thetype of motion used to aggregate pPSCs in general can be produced, forexample, by aerosolizing the vessel or chamber to produce a more laminarflow. The motion can also be created by having an inlet and an outletport to assist the inflow and outflow of the fluid medium, or even thecells themselves, to create motion similar to that achieved with theroller bottles described herein. The motion can also be achieved withthe use of one or more or a combination of flow distributors. Forexample, such a flow distributor may include a baffle to distribute theflow of fluid or medium within the chamber and thereby create acontinuous, uniform mixture of the three-dimensional cell aggregates. Inanother example, the flow distributor may be combination of one or moredeflector plates, distribution channels, and/or flow channels, whichcreate fluid movement similar to that found in roller bottles withoutnecessarily occurring in a roller bottle type, cylindrical vessel orchamber. Thus, alternative means of creating fluid movement in a mannerthat is non-turbulent, yet generates sufficient low shear force topromote cell collision and allow the cells to adhere to each other andform the cell aggregates as described herein.

Still, certain properties of growing adherent or anchorage dependentcells in roller bottles have their disadvantages. For example, adherentcell growth by its nature requires substantial surface area for the cellto attach to and roller bottles are limited in surface area that isavailable for growth. The conventional method of mixing in rollerbottles is rotation at a uniform rate in one direction for all purposese.g. cell planting or seeding, cell growth and/or virus propagation andexpansion. Standard rotation frequencies of most roller bottle processesfor culturing adherent and anchorage dependent cells is about 0.125 rpmto 5 rpm. For these cultures, it is important that the cells come intocontact with the sides of the roller bottle as rapidly as possible,since only after attachment to the vessel wall can the cellssubsequently proliferate and form cell sheets. Slow cell attachment tothe inner walls of the vessel leads to low viability of the cells and/orinhomogeneous planting, and hence inhomogeneous growth on the rollerbottle surface. Moreover, inefficient mixing limits cell growth becausethe cells do not obtain adequate nutrients (e.g. oxygen) or adequateremoval of toxins (e.g. carbon dioxide) from a submerged,surface-attached cell sheet as the bottle rotates. Interestingly, thesedisadvantages are not critical to using roller bottles for aggregation,growth, expansion and differentiation of differentiable pluripotentcells in suspension.

In view of the properties described above and further in viewApplicant's own disclosure of methods for making hES cell aggregates in6-well trays and the like, one of ordinary skill in the art would notturn to use of roller bottles for making pluripotent stem cellaggregates. See Schulz et al. 2012, Stem Cells 7: 1-17, e37004, and U.S.Pat. Nos. 8,153,429 and 8,008,075, which are incorporated herein byreference in their entireties. For example, Schulz et al. 2012, supra,teaches that pluripotent stem cells can be effectively aggregated byusing a circular or radial movement or motion or rotation, which isimposed over a central vortex and draws cells into a higher localdensity in the middle of the culture vessel, e.g. drawing cells into thecenter of a well of a 6-well tray, or the center of Erlenmeyer flask orthe center of a bioreactor based on a rotational format. This radialvortex cannot be accomplished in roller bottles because by its naturethe roller bottle rotates on its side wall and not on its base, hence itis not intuitive to transfer methods from a system that includes acentral vortex motion to one that does not, such as the roller bottlesas described herein.

Applicants have performed studies of static cultures using other typesof motion including studies rocking, stirring and centrifugation of hEScells, and these types of motions were incapable of allowing theformation of hES cell aggregates or differentiable cell aggregates.Further, these hES or hES cell-derived aggregates that did form underthese conditions did not give rise to functioning glucose responsivecell types in vivo, which is the ultimate test of any method forsuccessful manufacturing of PEC. See at least Kroon et al. 2008, supraand Schulz et al. (2012) supra. So, it cannot be said that just movementand motion alone is sufficient to form pluripotent stem cell or hES cellsuspension aggregates or differentiable cell aggregates, because it isnot. These studies (data not shown) indicated that more than just fluidmovement and forces generated with such movement facilitate the adhesivecontact necessary for cell aggregate formation that results in thetransitioning of single-cell pluripotent stem cells to stable cell-cellaggregates.

As mentioned briefly above, rotation of a roller bottle is verydifferent from rotation of a 6-well tray, Erlenmeyer flasks, and thelike which occurs about a central vortex. In a roller bottle, themajority of the culture volume remains at the bottom of the bottle whenthe bottle rotates on its side wall, and a thin layer of fluid orculture medium coats the inner bottle surface as the bottle rotates.This thin fluid layer has increased gas exchange as the bottle rotatesand therefore increases O₂ levels to the culture medium overall; i.e.once the thin layer of culture media returns to the bottom of the bottlewhere the majority of the culture medium resides, it carries with itincreases amounts of O₂ It is not intuitive then that this motion,especially when rotated at very low speeds that are standard in the artfor adherent cells (e.g. 0.125 to 5 rpm) would allow for sufficientcell-to-cell contact or collisions while at the same time maintain thelow shear-force sufficient to allow primate pluripotent stem cell (pPSC)aggregate formation, let alone differentiation of differentiable cellaggregates.

Using roller bottles to aggregate, grow, passage, expand anddifferentiate cells is also different from 6-well trays, Erlenmeyerflasks, bioreactors because of the different rotations speeds betweenthe two formats. 6-well trays, Erlenmeyer flasks, bioreactors and thelike for example use higher rotation speeds of about 80, 85, 90, 95,100, 105, 110, 115 and 120 rpm, which are required for at least thepurpose of preventing the cell aggregates from agglomerating or formingclusters or larger cell masses in culture. Note, that the agglomeratedcell clusters (e.g., large aggregates of 300 μm or more) are not to beconfused with the roughly spherical cell aggregates, which are smaller(about 100-200 μm) and uniform in size. In contrast, the aggregation,growth, passaging, expansion and differentiation of pPSCs in rollerbottles is performed at relatively low rotations speeds of about 3, 4,5, 6, 7, 8, 9, and 10 rpms. These lower rotation speeds do not createthe same degree of shear force which occurs in 6-well trays, Erlenmeyerflasks, bioreactors and the like, and in view of Applicant's previousexperience (see Schulz et al. 2012, supra), it was not expected thatcell aggregate formation would succeed under these conditions.

An advantage of using roller bottles to aggregate, grow, passage, expandand differentiate pluripotent stem cells over that of other cell culturevessels is that once optimized in the smallest roller bottle, themethodologies will work very similarly in larger bottles withoutadditional substantial invention. For example, by using longer bottleswith the same standard cross-section, but substantially larger capacity,or by using arrays of bottles, total culture mass can be scaled usingthe same bottle diameter, diameter/volume ratio and rotation speed. Anincrease in roller bottle length (scaling) does not affect the cellaggregation or differentiation processes. So, scaling of the cellprocess or manufacture from 490 cm² roller bottles (11.12 cm indiameter, 17.30 cm in length including cap) to 850 cm² roller bottles(11.63 cm in diameter, 27.36 cm in length including cap) to 1750 cm²roller bottles (11.73 cm in diameter, 53.16 cm in length including cap)or greater does not involve substantially or significantly modificationother than that described herein.

For at least the above reasons, scalability of cell manufacturing inroller bottles is different from the rotational platform systems of6-well trays, Erlenmeyer flasks, bioreactors and the like. For example,in order to achieve a 1 L pluripotent stem culture of 1×10⁶ cells/mL,about thirty (30) 6-well trays would be required. Stated in another way,instead of using eighty (80) E-well trays (total 480 wells), the skilledartisan would only need 4, 850 cm², roller bottles. The skilled artisanwill appreciate that less manipulation and labor that is needed forroller bottle culture is an improvement in manufacturing. In addition,adjustments must be made in volume, speed and rotational radius in orderto achieve aggregation when using rotational platforms at differentscales. Primate PSC aggregation in conical flasks can be achieved, forexample, but occurs best at relatively high rotation speeds, about 150rpm, and this causes too much turbulence and shear-force leading toincreased cell death (data not shown). Merely placing a bottle or jar ona rocking platform does not produce the herein described cell suspensionaggregates (data not shown). The rocking motion does not create asuitable fluid motion to support appropriate cell-cell contact andadherence, and potentially creates too much turbulence and shear-force,causing increased cell death. Similarly, square shaped bottles and 15 cmglass jars are unsuitable culture vessels to scale up pPSC aggregatesformation for similar reasons (data not shown). Further, merecell-to-cell contact alone does not cause pPSC aggregates to formbecause when cell pellets are recovered after single-cell suspensions ofhES cells are centrifuged, cell aggregates were not observed (data notshown). Thus, discovering a truly scalable system with appropriate fluidmotion that supports efficient and consistent cell aggregation(including consistent aggregate diameter) has not been at all straightforward or routine, and substantially more difficult than a skilledartisan would anticipate or expect. In fact, it has been surprising thatroller bottles, which have been traditionally used for large scale-upcultures of adherent and anchorage dependent cell types, coupled with upto a 30-fold reduction in speed would provide suitable conditions forproduction of pPSC aggregates and differentiation as herein described atall.

As used herein, the term “contacting” (i.e., contacting a cell e.g., adifferentiable cell, with a compound) is intended to include incubatingthe compound and the cell together in vitro (e.g., adding the compoundto cells in culture). The term “contacting” is not intended to includethe in vivo exposure of cells to a defined cell medium comprising anErbB3 ligand, and optionally, a member of the TGF-β family, that mayoccur naturally in a subject (i.e., exposure that may occur as a resultof a natural physiological process). The step of contacting the cellwith a defined cell medium comprising an ErbB3 ligand, and optionally, amember of the TGF-β family, can be conducted in any suitable manner. Forexample, the cells may be treated in adherent culture, or in suspensionculture. It is understood that the cells contacted with the definedmedium can be further treated with a cell differentiation environment tostabilize the cells, or to differentiate the cells.

As used herein, the term “differentiate” refers to the production of acell type that is more differentiated than the cell type from which itis derived. The term therefore encompasses cell types that are partiallyand terminally differentiated. Differentiated cells derived from hESCare generally referred to as hES-derived cells or hES-derived cellaggregate cultures, or hES-derived single cell suspensions, orhES-derived cell adherent cultures and the like.

As used herein, the term “substantially” refers to a great extent ordegree, e.g. “substantially similar” in context is used to describe onemethod which is to great extent or degree similar to or different thananother method. However, as used herein, the term “substantially free”,e.g., “substantially free” or “substantially free from contaminants,” or“substantially free of serum” or “substantially free of insulin orinsulin like growth factor” or equivalents thereof, means that thesolution, media, supplement, excipient and the like, is at least 98%, orat least 98.5%, or at last 99%, or at last 99.5%, or at least 100% freeof serum, contaminants or equivalent thereof. In one embodiment, thereis provided a defined culture media with no serum, or 100% serum-free,or substantially free of serum. Conversely, as used herein, the term“substantially similar” or equivalents thereof means that thecomposition, process, method, solution, media, supplement, excipient andthe like is at least 80%, at least 85%, at least 90%, at least 95%, orat least 99% similar to that previously described in the specificationherein, or in a previously described process or method incorporatedherein in its entirety.

In certain embodiments of the present invention, the term “enriched”refers to a cell culture that contains more than approximately 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the desired cell lineage.

As used herein, the term “effective amount” or equivalents thereof of acompound refers to that concentration of the compound that is sufficientin the presence of the remaining components of the defined medium toeffect the stabilization of the differentiable cell in culture forgreater than one month in the absence of a feeder cell and in theabsence of serum or serum replacement. This concentration is readilydetermined by one of ordinary skill in the art.

As used herein, the term “express” refers to the transcription of apolynucleotide or translation of a polypeptide in a cell, such thatlevels of the molecule are measurably higher in a cell that expressesthe molecule than they are in a cell that does not express the molecule.Methods to measure the expression of a molecule are well known to thoseof ordinary skill in the art, and include without limitation, Northernblotting, RT-PCR, in situ hybridization, Western blotting, andimmunostaining

As used herein when referring to a cell, cell line, cell culture orpopulation of cells, the term “isolated” refers to being substantiallyseparated from the natural source of the cells such that the cell, cellline, cell culture, or population of cells are capable of being culturedin vitro. In addition, the term “isolating” is used to refer to thephysical selection of one or more cells out of a group of two or morecells, wherein the cells are selected based on cell morphology and/orthe expression of various markers.

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein. However, before the presentcompositions and methods are disclosed and described, it is to beunderstood that this invention is not limited to specific nucleic acids,specific polypeptides, specific cell types, specific host cells,specific conditions, or specific methods, etc., as such may, of course,vary, and the numerous modifications and variations therein will beapparent to those skilled in the art.

Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described inSambrook et al. 1989 “Molecular Cloning”, Second Edition, Cold SpringHarbor Laboratory, Plainview, N.Y.; Maniatis et al. 1982 “MolecularCloning”, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) 1993Meth. Enzymol. 218, Part I; Wu (ed.) 1979 Meth. Enzymol. 68; Wu et al.(eds.) 1983 Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) 1980Meth. Enzymol. 65; Miller (ed.) 1972 “Experiments in MolecularGenetics”, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Oldand Primrose, 1981 “Principles of Gene Manipulation”, University ofCalifornia Press, Berkeley; Schleif & Wensink, 1982 “Practical Methodsin Molecular Biology”; Glover (ed.) 1985 “DNA Cloning” Vol. I and II,IRL Press, Oxford, UK; Hames and Higgins (eds.) 1985 “Nucleic AcidHybridization”, IRL Press, Oxford, UK; and Setlow and Hollaender 1979“Genetic Engineering: Principles and Methods”, Vols. 1-4, Plenum Press,New York. Abbreviations and nomenclature, where employed, are deemedstandard in the field and commonly used in professional journals such asthose cited herein.

The invention relates to compositions and methods comprising a basalsalt nutrient solution and an effective amount of an ErbB3 ligand, withthe compositions being essentially free of serum. The compositions andmethods of the present invention are useful for culturing cells, inparticular, differentiable cells. It is understood that at differentpoints during culturing the differentiable cells, various components maybe added to the cell culture such that the medium can contain componentsother than those described herein. It is, however, contemplated that atleast at one point during the preparation of the culture, or during theculture of the differentiable cells, the defined medium comprises abasal salt nutrient solution and a means for activating ErbB2-directedtyrosine kinase.

Although a basal salt nutrient solution as described herein is employedto maintain cell growth and viability of hESC, in other embodiments ofthe invention, alternative stem cell culture medias to maintainpluripotency or for differentiation of the pluripotent cells, work insubstantially similar means, including but not limited to KSR(Invitrogen), or xeno-free KSR (Invitrogen), StemPro® (Invitrogen),mTeSR™1 (StemCell Technologies) and HEScGRO (Millipore), DMEM basedmedia, and the like.

In another embodiment, hESC are cultured in the defined media describedherein in the absence and/or presence of extracellular matrix proteins(ECM), e.g., MATRIGEL. Human ES cells cultured in the absence of ECMcontain about 0.5 to 10% human serum (hS) or hS retentate fractions froma 300K and/or 100K cut-off spin column (Microcon). The hES cellaggregate suspensions can be produced by directly incubating the hESCinto the media containing hS or hS retentate fractions; or afterincubating the culture vessels with the hS or hS retentate fractions forabout 30 min., 1 hour, 2 hours, 3 hours, 4, hours, 5 hours, 6 hours, 12hours, and 24 hours at 37° C. The plating efficiency for the hESC in thehS or hS retentate fraction containing media was comparable to thatobserved in hESC cultured in DC-HAIF as described in PCT/US2007/062755,or cultured in DC-HAIF media using MATRIGEL™ as an ECM, or other similarmatrices. Methods for culturing hESC in a defined media substantiallyfree of serum is described in U.S. patent application Ser. No.11/8875,057, filed Oct. 19, 2007, entitled METHODS AND COMPOSITIONS FORFEEDER-FREE PLURIPOTENT STEM CELL MEDIA CONTAINING HUMAN SERUM, which isherein incorporated in its entirety by reference.

Still in another embodiment, hES cell aggregate suspensions werecultured in a media substantially free of serum and further in theabsence of exogenously added fibroblast growth factor (FGF). This isdistinguished from U.S. Pat. No. 7,005,252 (Thomson), which requiresculturing hESC in a media without serum but containing exogenously addedgrowth factors, including FGF.

Cellular regulation can be effected through the transduction ofextracellular signals across the membrane that, in turn, modulatesbiochemical pathways within the cell. Protein phosphorylation representsone course by which intracellular signals are propagated from moleculeto molecule resulting finally in a cellular response. These signaltransduction cascades are highly regulated and often overlapping asevidenced by the existence of many protein kinases as well asphosphatases. It has been reported that in humans, protein tyrosinekinases are known to have a significant role in the development of manydisease states including diabetes, cancer and have also been linked to awide variety of congenital syndromes. Serine threonine kinases, e.g.,Rho kinases, are a class of enzymes, which if inhibited can haverelevance to the treatment of human disease, including diabetes, cancer,and a variety of inflammatory cardiovascular disorders and AIDS. Themajority of inhibitors identified/designed to date act at theATP-binding site. Such ATP-competitive inhibitors have demonstratedselectivity by virtue of their ability to target the more poorlyconserved areas of the ATP-binding site.

The Rho kinase family of small GTP binding proteins contains at least 10members including Rho A-E and G, Rac 1 and 2, Cdc42, and TC10. Theinhibitors are often referred to as ROK or ROCK inhibitors, and they areused interchangeably herein. The effector domains of RhoA, RhoB, andRhoC have the same amino acid sequence and appear to have similarintracellular targets. Rho kinase operates as a primary downstreammediator of Rho and exists as two isoforms: α (ROCK2) and β (ROCK1). Rhokinase family proteins have a catalytic (kinase) domain in theirN-terminal domain, a coiled-coil domain in their middle portion, and aputative pleckstrin-homology (PH) domain in their C-terminal domain. TheRho-binding domain of ROCK is localized in the C-terminal portion of thecoiled-coil domain and the binding the GTP-bound form of Rho results inenhancement of kinase activity. The Rho/Rho-kinase-mediated pathwayplays an important role in the signal transduction initiated by manyagonists, including angiotensin II, serotonin, thrombin, endothelin-1,norepinephrine, platelet-derived growth factor, ATP/ADP andextracellular nucleotides, and urotensin II. Through the modulation ofits target effectors/substrates Rho kinase plays an important role invarious cellular functions including smooth muscle contraction, actincytoskeleton organization, cell adhesion and motility and geneexpression. By virtue of the role that Rho kinase protein play inmediating a number of cellular functions perceived to be associated withthe pathogenesis of arteriosclerosis, inhibitors of this kinase may alsobe useful for the treatment or prevention of various arterioscleroticcardiovascular diseases and involved in endothelial contraction andenhancement of endothelial permeability which is thought to progress toatherosclerosis. Hence, in other embodiments of the invention, agentswhich promote and/or support cell survival are added to various cellculture media, for example, Rho-kinase inhibitors Y-27632, Fasudil, andH-1152P and ITS (insulin/transferrin/selenium; Gibco). These cellsurvival agents function, in part, by promoting re-association ofdissociated hES cell or hES-derived cultures, e.g., foregut endoderm,pancreatic endoderm, pancreatic epithelium, pancreatic progenitorpopulations and the like, particularly dissociated pancreatic endodermand pancreatic progenitor populations. Increase in survival of hES orhES-derived cells was achieved independently of whether the cells wereproduced from cell aggregates in suspension or from adherent platecultures (with or with no extracellular matrix, with or without serum,with or without feeders). Increase in survival of these cell populationsfacilitates and improves purification systems using a cell-sorter and,therefore allows improved recovery of the cells. Use of Rho kinaseinhibitors such as Y27632 may allow for expansion of hES-derived celltypes as well by promoting their survival during serial passagingdissociated single cells or from cryogenic preservation. Although, Rhokinase inhibitors such as Y27632 have been tested on hES and hES-derivedcell cultures, Rho kinase inhibitors can be applied to other cell types,for example, in general, epithelial cells including but not limited tointestinal, lung, thymus, kidney as well as neural cell types likepigmented retinal epithelium.

As used herein, the term “differentiable cell” is used to describe acell or population of cells that can differentiate into at leastpartially mature cells, or that can participate in the differentiationof cells, e.g., fuse with other cells, that can differentiate into atleast partially mature cells. As used herein, “partially mature cells”,“progenitor cells”, “immature cells”, “precursor cells”, “multipotentcells” or equivalents thereof and also includes those cells which areterminally differentiated, e.g., definitive endoderm cells,PDX1-negative foregut endoderm cells, PDX1-positive pancreatic endodermcells which further include PDX1-positive pre-pancreatic endoderm cellsand PDX1-positive pancreatic endoderm tip cells. All are cells thatexhibit at least one characteristic of the phenotype, such as morphologyor protein expression, of a mature cell from the same organ or tissuebut can further differentiate into at least one other cell type. Forexample, a normal, mature hepatocyte typically expresses such proteinsas albumin, fibrinogen, alpha-1-antitrypsin, prothrombin clottingfactors, transferrin, and detoxification enzymes such as the cytochromeP-450s, among others. Thus, as defined in the present invention, a“partially mature hepatocyte” may express albumin or another one or moreproteins, or begin to take the appearance or function of a normal,mature hepatocyte.

In contrast to cell aggregates produced by previously known methods thatmay vary in both size and shape, the cell aggregates and methodsdescribed herein have a narrow size and shape distribution, i.e., thecell aggregates are substantially uniform in size and/or shape. The sizeuniformity of the cell aggregates is critical for differentiationperformance and homogeneity of the culture. Applying basic masstransport analysis to the aggregates, it is expected that diffusion ofoxygen and nutrients into the center of large aggregates will be slowcompared to diffusion into smaller aggregates, assuming equalpermeability. As differentiation of aggregated ES cells into pancreaticlineage cells is dependent on the temporal application of specificgrowth factors, a culture with a mixture of aggregates of differentdiameters is likely to be de-synchronized as compared to a uniform (sizeand shape) culture of cell aggregates. This mixture of cell aggregatesgives rise to heterogeneity and may result in poor differentiationperformance and ultimately not lend itself to being amenable tomanufacturing, scale-up, and production. The cell aggregates used hereincan be of various shapes, such as, for example, a sphere, a cylinder(preferably with equal height and diameter), or rod-like among others.Although other shaped aggregates may be used, in one embodiment of theinvention, it is generally preferable that the cell aggregates bespherical or cylindrical. In another embodiment, the cell aggregates arespherical and substantially uniform in size and shape. For instance, ifthe cell aggregates differ in size or are not uniform, it will bedifficult to reliably manufacture and perform large scale-up processesof the cells. Hence, as used herein, the phrase “substantially uniform”or “substantially uniform in size and shape” or equivalents thereof,refers to the spread in uniformity of the aggregates which is not morethan about 20%. In another embodiment, the spread in uniformity of theaggregates is not more than about 15%, 10% or 5%.

Although the exact number of cells per aggregate is not critical, itwill be recognized by those skilled in the art that the size of eachaggregate (and thus the number of cells per aggregate) is limited by thecapacity of oxygen and nutrients to diffuse to the central cells, andthat this number may also vary depending on cell type and the nutritiverequirements of that cell type. Cell aggregates may comprise a minimalnumber of cells (e.g., two or three cells) per aggregate, or maycomprise many hundreds or thousands of cells per aggregate. Typically,cell aggregates comprise hundreds to thousands of cells per aggregate.For purposes of the present invention, the cell aggregates are typicallyfrom about 50 microns to about 600 microns in size, although, dependingon cell type, the size may be less or greater than this range. In oneembodiment, the cell aggregates are from about 50 microns to about 250microns in size, or about 75 to 200 microns in size, and preferably theyare about 100 to 150 microns in size. In contrast, cylindrical ornon-spherical cell aggregates which may occur in suspension are thoseaggregates whereby the diameter, as based on the minor and major axes(e.g., X, Y and Z), are not equal. These non-spherical cell aggregatestend to be larger in size, about 500 microns to 600 microns in diameterand height. However, in the methods described herein, thesenon-spherical hES cell aggregates become spherical once differentiationis initiated if they were not already. Non-spherical cell aggregatesinclude but are not limited to cylindrical and cuboidal cell aggregates,but are still uniform in size and shape.

Many cell types may be used to form the cell aggregates describedherein. In general, the choice of cell type will vary depending on thetype of three-dimensional construct to be engineered (e.g. various organstructures including pancreas, liver, lung, kidney, heart, bladder,blood vessels, and the like). For example, if the three dimensionalstructure is a pancreas, the cell aggregates will advantageouslycomprise a cell type or types typically found in a pancreas (e.g.,endocrine cells such as insulin, glucagon, ghrelin, somatostatin typecells, as well as endothelial cells, smooth muscle cells, etc.). Oneskilled in the art can choose an appropriate cell type(s) for the cellaggregates, based on the type of three-dimensional tissue or organ to bedesired. Non-limiting examples of suitable cell types include stem cells(e.g. adult and embryonic), contractile or muscle cells (e.g., striatedmuscle cells and smooth muscle cells), neural cells (e.g., glial,dendritic and neurons), connective tissue (including bone, cartilage,cells differentiating into bone forming cells and chondrocytes, andlymph tissues), parenchymal cells, epithelial cells (includingendothelial cells that form linings in cavities and vessels or channels,exocrine secretory epithelial cells, epithelial absorptive cells,keratinizing epithelial cells (e.g. keratinocytes and corneal epithelialcells), extracellular matrix secretion cells, mucosal epithelial cells,renal epithelial cells, lung epithelial cells, mammary epithelial cellsand the like, and undifferentiated cells (such as embryonic cells, stemcells, and other precursor cells), among others.

The cell aggregates described herein can be homo-cellular aggregates orhetero-cellular aggregates. As used herein, “homo-cellular”,“mono-cellular” cell aggregates or equivalents thereof refers to aplurality of cell aggregates in suspension, wherein each cell aggregatecomprises a plurality of living cells of substantially a single celltype, e.g. methods for producing hES cell aggregates described hereincan be substantially homo-cellular, consisting substantially ofpluripotent hESC, consisting of substantially of definitive endodermcells, foregut endoderm cells, consisting substantially of pancreaticendoderm cells, which can further include PDX1-positive pre-pancreaticendoderm cells, PDX1-positive pancreatic endoderm cells, PDX1-positivepancreatic endoderm tip cells, pancreatic endocrine precursor cells,pancreatic endocrine cells and the like.

As used herein, the term “essentially” or “substantially” means either ade minimus or a reduced amount of a component or cell present in anycell aggregate suspension type, e.g., cell aggregates in suspensiondescribed herein are “essentially or substantially homogenous”,“essentially or substantially homo-cellular” or are comprised of“essentially hESC”, “essentially or substantially definitive endodermcells”, “essentially or substantially foregut endoderm cells”,“essentially or substantially PDX1-negative foregut endoderm cells”,“essentially or substantially PDX1-positive pre-pancreatic endodermcells”, “essentially or substantially PDX1-positive pancreatic endodermor progenitor cells”, “essentially or substantially PDX1-positivepancreatic endoderm tip cells”, “essentially or substantially pancreaticendocrine precursor cells”, “essentially or substantially pancreaticendocrine cells” and the like.

Some of the substantially homo-cellular cell aggregate suspensioncultures are, for example, hES-derived cell aggregate suspensioncultures which comprise less than about 50% hESCs, less than about 45%hESCs, less than about 40% hESCs, less than about 35% hESCs, less thanabout 30% hESCs, less than about 25% hESCs, less than about 20% hESCs,less than about 15% hESCs, less than about 10% hESCs, less than about 5%hESCs, less than about 4% hESCs, less than about 3% hESCs, less thanabout 2% hESCs or less than about 1% hESCs of the total hES-derivedcells in the culture. Stated in another way, hES-derived cell aggregatesuspension cultures, e.g., PDX1-negative foregut endoderm, PDX-positivepre-pancreatic endoderm cells, PDX1-positive pancreatic endoderm orprogenitor cells, PDX1-positive pancreatic tip cells, pancreaticendocrine progenitor cells and pancreatic endocrine cells, comprise atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%,

As used herein, “hetero-cellular”, “multi-cellular” or equivalentsthereof refers to cell aggregates whereby each individual cell aggregatecomprises a plurality of cells of at least two, three, four, five, sixor more cell types, or at least one cell type and a non-cellularcomponent, e.g., extracellular matrix (ECM) material (e.g., collagen,fibronectin, laminin, elastin, and/or proteoglycans). Such ECMcomponents can be naturally secreted by the cells, or alternately, thecells can be genetically manipulated by any suitable method known in theart to vary the expression level of ECM material and/or cell adhesionmolecules, such as selectins, integrins, immunoglobulins, and cadherins,among others. In another embodiment, either natural ECM material or anysynthetic component that imitates ECM material can be incorporated intothe aggregates during aggregate formation. For example, methods forproduction of hES-derived cell aggregates such as pancreatic epithelialor pancreatic endoderm cell aggregates (or stage 4 cell aggregates)described herein consists substantially of pancreatic epithelial orendoderm cells, but may also consist in small cell numbers othernon-pancreatic epithelial type cells, or other endoderm progenitors, andeven pancreatic endocrine secreting cells (e.g., insulin secretingcells).

To be clear, the homo- or hetero-cellular aggregates described hereinand produced by the suspension methods described herein, are not thesame cell aggregates described in the art and by others and referred toas embryoid bodies (EBs). Embryoid bodies are clearly distinguished fromthe herein described cell aggregates because EBs are cell aggregates ofdifferentiated and undifferentiated cells that appear when ES cellsovergrow in monolayer cultures, or are maintained in suspension culturesin undefined media or are differentiated via non-directed protocols(i.e. random differentiation) towards multiple germ layer tissues. Incontrast, the present invention, discussed in detail in Examples 17 and20, enzymatically dissociates hESC on adherent plate cultures to make asingle cell suspension and then brings the cells together to form cellaggregates; then using these cell aggregates suspension cultures fordifferentiation substantially as described in D'Amour et al. 2005,supra, & D'Amour et al. 2006, supra. Other differences between EBs andthe cell aggregates of this invention are further discussed below.

Still other methods describe making embryoid bodies (EBs). As usedherein, the term “embryoid bodies”, “aggregate bodies” or equivalentsthereof, refer to aggregates of differentiated and undifferentiatedcells that appear when ES cells overgrow in monolayer cultures, or aremaintained in suspension cultures in undefined media or aredifferentiated via non-directed protocols towards multiple germ layertissues. That is, EBs are not formed from a single cell suspension ofpluripotent stem cells as described herein; nor are EBs formed fromadherent cultures of hES-derived multipotent cells. These features alonemake the present invention clearly distinguishable from an embryoidbody.

Embryoid bodies are a mixture of different cell types, typically fromseveral germ layers, distinguishable by morphological criteria. Embryoidbodies typically refer to a morphological structure comprised of apopulation of cells, the majority of which are derived from embryonicstem (ES) cells that have undergone non-directed differentiation, i.e.,such as that which occurs when undifferentiated cells are exposed tohigh concentrations of serum in the absence of defined growth factors.Under culture conditions suitable for EB formation (e.g., the removal ofLeukemia inhibitory factor for mouse ES cells, or other, similarblocking factors), ES cells proliferate and form small masses of cellsthat begin to differentiate. First, corresponding to about days 1-4 ofdifferentiation for human ES cells, the small mass of cells forms alayer of endodermal cells on the outer layer, and is considered a“simple embryoid body”. Secondly, corresponding to about days 3-20 postdifferentiation for human ES cells, “complex embryoid bodies” areformed, which are characterized by extensive differentiation ofectodermal and mesodermal cells and derivative tissue. As used herein,EBs includes both simple and complex EBs unless otherwise required bycontext. The determination of when embryoid bodies have formed in aculture of ES cells is routinely made by persons of skill in the art by,for example, visual inspection of the morphology. Floating masses ofabout 20 cells or more depending on the culture conditions areconsidered to be EBs. See, e.g., Schmitt et al. (1991) Genes Dev. 5,728-740; Doetschman et al. 1985, J. Embryol. Exp. Morph. 87:27-45. Theterm also refers to equivalent structures derived from primordial germcells, which are primitive cells extracted from embryonic gonadalregions; see e.g., Shamblott et al. 1998, Proc. Natl. Acad. Sci. USA 95:13726. Primordial germ cells, sometimes also referred to in the art asEG cells or embryonic germ cells, when treated with appropriate factorsform pluripotent ES cells from which embryoid bodies can be derived; seee.g., U.S. Pat. No. 5,670,372; and Shamblott et al. supra.

Various methods for making EBs exist, e.g. spin embryoid bodies asdescribed by Ng et al. 2008 Nature Protocols 3:468-776 and EBs made fromsingle cell suspensions which were plated onto micro-patternedextracellular matrix islands as described in Bauwens et al. 2008, StemCells 26:2300-10, Epub 2008 Jun. 26. However, these methods arecost-prohibitive and less efficient for large scaled production(manufacturing) of hESC and hES-derived cells because they require toomany steps before scale-up production can actually commence. Forexample, Bauwens et al. first have to seed hESC on a growth factorreduced MATRIGEL™ before the cells can be selected to start a suspensionculture. The time and cost of this method makes it cumbersome becausecustomized micro-patterned tissue culture plates are required.Additionally, the method employed by Ng et al. is also notcost-efficient for large scale-up manufacturing of hESC and hES-derivedcells because of the use of centrifuges in order to create a moreuniform EB. These methods are limited by surface area constraints, whichalso impacts their scalability. Lastly, in all these methodologies, thecell aggregates are not made from single cell suspensions of pluripotentstem cells as in the present invention.

Embryoid bodies are cell aggregates, unlike the cell aggregatesdescribed in this invention, that are made up of numerous cell typesfrom the three germ layers and are typically created by exposingaggregates of undifferentiated ES cells to non-directed differentiationsignals, such as 20% fetal bovine serum. The result of this non-directedmethodology is a mixture of cell types that is intended to mimic normalembryo development in vitro. While this approach is useful at the basicresearch level for examining embryo development, it is not amenable toany large-scale cell therapy manufacturing process where cell yield,population identity, population purity, batch consistency, safety, cellfunction and cost of goods are primary concerns. Moreover, regardless ofany enrichment strategies employed to purify a given cell type from anembryoid body, the differentiation protocol does not provide a directedapproach that will generate a large population of a single cell type.Subsequently, contaminant populations will always predominate and willhamper any attempt to purify a specific population. All previous work oncreating and differentiating aggregates of ES cells has one or more ofthe following components in their methodology: 1) use of mouse ratherthan human ES cells, 2) forced aggregation protocols that rely oncentrifugation to aggregate cells rather than normal cell adhesionprocesses, 3) aggregation of cell chunks in static conditions, 4)non-single cell dissociation or scraping of cells off surfaces to createaggregates, 5) non-direct differentiation of cell aggregates using15-20% fetal calf serum, resulting in the formation of an embryoid bodyand cell types of all germ layers, 6) formation in “hanging drop”conditions that can only be performed at a small scale. To ourknowledge, the only study that does not utilize 15-20% FCS todifferentiate embryoid bodies describes a protocol where cell aggregatesare formed by forced aggregation, then aggregates are immediatelydifferentiated using media appropriate for mesoderm (Ng et al. 2005,Blood 106:1601). However, in this work, the researchers transferred theembryoid bodies to non-aggregate adherent culture after 10-12 days instatic aggregate culture making comparisons to the current applicationirrelevant. In contrast to all previous work, the current applicationpresents an approach that 1) dissociates human ES cells to single cellsthen creates aggregates by rotational culture at shear rates optimizedfor improve control of aggregate diameter and cell survival, 2) directlydifferentiates the ES cell aggregates to definitive endoderm thenforegut endoderm, then pre-pancreatic foregut endoderm, then pancreaticendoderm and finally pancreatic endocrine cells. This differentiationprotocol generates definitive endoderm and pancreatic lineagepopulations with high efficiency and minimal contaminant populations.Moreover, this approach to ES cell aggregation and differentiation doesnot create embryoid bodies, in direct contrast to all other publishedresearch.

In contrast to embryoid bodies, which are a mixture of differentiatedand undifferentiated cells and typically consist of cells from severalgerm layers and go through random differentiation, the cell aggregatesdescribed herein are essentially or substantially homo-cellular,existing as aggregates of pluripotent, multipotent, bipotent, orunipotent type cells, e.g., embryonic cells, definitive endoderm,foregut endoderm, PDX1 positive pancreatic endoderm, pancreaticendocrine cells and the like.

The present invention addresses the above problems by providing a costefficient manufacturing process or methods capable of reproduciblyproducing cell aggregates that are substantially uniform in size andshape using a process that can easily be applied to large scalemanufacturing. In one particular embodiment, the differentiable cellsare expanded in a suspension culture, using the cell media of thepresent invention. In another particular embodiment, the differentiablecells can be maintained and expanded in suspension, i.e., they remainundifferentiated or are prevented from further differentiating. The term“expand” in the context of cell culture is used as it is in the art, andrefers to cellular proliferation and increase the number of cells,preferably increase in number of viable cells. In a specific embodiment,the cells are expanded in a culture suspension by culturing for morethan about one day, i.e., about 24 hours. In a more specific embodiment,the cells are expanded in a suspension culture by culturing for at least1, 2, 3, 4, 5, 6, 7 days, or at least 2, 3, 4, 5, 6, 7, 8 weeks.

The differentiation culture conditions and hES-derived cell typesdescribed herein are substantially similar to that described in D'Amouret al. 2006, supra. D'Amour et al. 2006, describes a 5 stepdifferentiation protocol: stage 1 (results in substantially definitiveendoderm production), stage 2 (results in substantially PDX1-negativeforegut endoderm production), stage 3 (results in substantiallyPDX1-positive foregut endoderm production), stage 4 (results insubstantially pancreatic endoderm or epithelium or pancreatic endocrineprogenitor production) and stage 5 (results in substantially hormoneexpressing endocrine cell production). Importantly, for the first time,all these cell types can be produced by suspension methods describedherein.

As used herein, “definitive endoderm (DE)” refers to a multipotentendoderm lineage cell that can differentiate into cells of the gut tubeor organs derived from the gut tube. In accordance with certainembodiments, the definitive endoderm cells are mammalian cells, and in apreferred embodiment, the definitive endoderm cells are human cells. Insome embodiments of the present invention, definitive endoderm cellsexpress or fail to significantly express certain markers. In someembodiments, one or more markers selected from SOX17, CXCR4, MIXL1,GATA4, HNF3beta, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1, CRIP1 and CERare expressed in definitive endoderm cells. In other embodiments, one ormore markers selected from OCT4, alpha-fetoprotein (AFP), Thrombomodulin(TM), SPARC, SOX7 and HNF4alpha are not significantly expressed indefinitive endoderm cells. Definitive endoderm cell populations andmethods of production thereof are also described in U.S. patentapplication Ser. No. 11/021,618, entitled DEFINITIVE ENDODERM, filedDec. 23, 2004, which is hereby incorporated in its entirety.

Still other embodiments of the present invention relate to cell culturesand cell aggregates termed “PDX1-negative foregut endoderm cells”,“foregut endoderm cells” or equivalents thereof. PDX1-negative foregutendoderm cells are also multipotent and can give rise to various cellsand tissues including but not limited to thymus, thyroid, parathyroid,lungs/bronchi, liver, pharynx, pharyngeal pouches, parts of the duodenumand Eustachian tube. In some embodiments, the foregut endoderm cellsexpress increased levels of SOX17, HNF1B, HNF1 alpha, FOXA1 as comparedto non foregut endoderm cells e.g., definitive endoderm or PDX-positiveendoderm which do not appreciably express these markers. PDX1-negativeforegut endoderm cells also express low to no levels of PDX1, AFP, SOX7and SOX1. PDX1-negative foregut endoderm cell populations and methods ofproduction thereof are also described in U.S. patent application Ser.No. 11/588,693, entitled PDX1-expressing dorsal and ventral foregutendoderm, filed Oct. 27, 2006 which is hereby incorporated herein byreference in its entirety.

Other embodiments of the present invention relate to cell cultures of“PDX1-positive pancreatic foregut endoderm cells,” “PDX1-positivepre-pancreatic endoderm,” or equivalents thereof. PDX1-positivepre-pancreatic endoderm cells are multipotent and can give rise tovarious cells and/or tissues including but not limited to stomach,intestine and pancreas. In some embodiments, the PDX1-positivepre-pancreatic endoderm cells express increased levels of PDX1, HNF6,SOX9 and PROX1 as compared to non pre-pancreatic endoderm cells which donot appreciably express these markers. PDX1-positive pre-pancreaticendoderm cells also express low to no levels of NKX6.1, PTF1A, CPA, andcMYC.

Other embodiments of the present invention relate to cell cultures of“PDX1-positive pancreatic endoderm cells,” “PDX1-positive pancreaticprogenitor,” “pancreatic epithelium”, “PE” or equivalents thereof.PDX1-positive pancreatic progenitor cells are multipotent and can giverise to various cells in the pancreas including but not limited toacinar, duct and endocrine cells. In some embodiments, the PDX1-positivepancreatic progenitor cells express increased levels of PDX1 and NKX6.1as compared to non pre-pancreatic endoderm cells which do notappreciably express these markers. PDX1-positive pancreatic progenitorcells also express low to no levels of PTF1A, CPA, cMYC, NGN3, PAX4, ARXand NKX2.2, INS, GCG, GHRL, SST, and PP.

Alternatively, other embodiments of the present invention relate to cellcultures of “PDX1-positive pancreatic endoderm tip cells,” orequivalents thereof. In some embodiments, the PDX1-positive pancreaticendoderm tip cells express increased levels of PDX1 and NKX6.1 similarto PDX1-positive pancreatic progenitor cells, but unlike PDX1-positivepancreatic progenitor cells, PDX1-positive pancreatic endoderm tip cellsadditionally express increased levels of PTF1A, CPA and cMYC.PDX1-positive pancreatic endoderm tip cells also express low to nolevels of NGN3, PAX4, ARX and NKX2.2, INS, GCG, GHRL, SST, and PP.

Yet, other embodiments of the present invention relate to cell culturesof “pancreatic endocrine precursor cells,” “pancreatic endocrineprogenitor cells” or equivalents thereof. Pancreatic endocrineprogenitor cells are multipotent and give rise to mature endocrine cellsincluding alpha, beta, delta and PP cells. In some embodiments, thepancreatic endocrine progenitor cells express increased levels of NGN3,PAX4, ARX and NKX2.2 as compared to other non-endocrine progenitor celltypes. Pancreatic progenitor cells also express low to no levels of INS,GCG, GHRL, SST, and PP.

Still other embodiments of the present invention relate to cell culturesof “pancreatic endocrine cells,” “pancreatic hormone secreting cells”,“pancreatic islet hormone-expressing cell,” or equivalents thereof thatrefer to a cell, which has been derived from a pluripotent cell invitro, e.g. alpha, beta, delta and/or PP cells or combinations thereof.The endocrine cells can be poly-hormonal or singly-hormonal, e.g.expressing insulin, glucagon, ghrelin, somatostatin and pancreaticpolypeptide or combinations thereof. The endocrine cells can thereforeexpress one or more pancreatic hormones, which have at least some of thefunctions of a human pancreatic islet cell. Pancreatic islethormone-expressing cells can be mature or immature. Immature pancreaticislet hormone-expressing cells can be distinguished from maturepancreatic islet hormone-expressing cells based on the differentialexpression of certain markers, or based on their functionalcapabilities, e.g., glucose responsiveness in vitro or in vivo.Pancreatic endocrine cells also express low to no levels of NGN3, PAX 4,ARX and NKX2.2.

Most of above cell types are epithelialized as compared to mesenchymaldefinitive endoderm cells. In some embodiments, the pancreatic endodermcells express one or more markers selected from Table 3 and/or one ormore markers selected from Table 4 of related U.S. patent applicationSer. No. 11/588,693 entitled PDX1 EXPRESSING DOSAL AND VENTRAL FOREGUTENDODERM, filed Oct. 27, 2006, and also U.S. patent application Ser. No.11/115,868, entitled PDX1-expressing endoderm, filed Apr. 26, 2005,which are hereby incorporated herein by reference in their entireties.

The invention contemplates compositions and methods useful fordifferentiable cells, regardless of their source or of their plasticity.The “plasticity” of a cell is used herein roughly as it is in the art.Namely, the plasticity of a cell refers to a cell's ability todifferentiate into a particular cell type found in tissues or organsfrom an embryo, fetus or developed organism. The “more plastic” a cell,the more tissues into which the cell may be able to differentiate.“Pluripotent cells” include cells and their progeny, which may be ableto differentiate into, or give rise to, pluripotent, multipotent,oligopotent and unipotent cells, and/or several, if not all, of themature or partially mature cell types found in an embryo, fetus ordeveloped organism. “Multipotent cells” include cells and their progeny,which may be able to differentiate into, or give rise to, multipotent,oligopotent and unipotent progenitor cells, and/or one or more mature orpartially mature cell types, except that the mature or partially maturecell types derived from multipotent cells are limited to cells of aparticular tissue, organ or organ system. For example, a multipotenthematopoietic progenitor cell and/or its progeny possess the ability todifferentiate into or give rise to one or more types of oligopotentcells, such as myeloid progenitor cells and lymphoid progenitor cells,and also give rise to other mature cellular components normally found inthe blood. “Oligopotent cells” include cells and their progeny whoseability to differentiate into mature or partially mature cells is morerestricted than multipotent cells. Oligopotent cells may, however, stillpossess the ability to differentiate into oligopotent and unipotentcells, and/or one or more mature or partially mature cell types of agiven tissue, organ or organ system. One example of an oligopotent cellis a myeloid progenitor cell, which can ultimately give rise to matureor partially mature erythrocytes, platelets, basophils, eosinophils,neutrophils and monocytes. “Unipotent cells” include cells and theirprogeny that possess the ability to differentiate or give rise to otherunipotent cells and/or one type of mature or partially mature cell type.

Differentiable cells, as used herein, may be pluripotent, multipotent,oligopotent or even unipotent. In certain embodiments of the presentinvention, the differentiable cells are pluripotent differentiablecells. In more specific embodiments, the pluripotent differentiablecells are selected from the group consisting of embryonic stem cells,ICM/epiblast cells, primitive ectoderm cells, primordial germ cells, andteratocarcinoma cells. In one particular embodiment, the differentiablecells are mammalian embryonic stem cells. In a more particularembodiment, the differentiable cells are human embryonic stem cells.

The invention also contemplates differentiable cells from any sourcewithin an animal, provided the cells are differentiable as definedherein. For example, differentiable cells may be harvested from embryos,or any primordial germ layer therein, from placental or chorion tissue,or from more mature tissue such as adult stem cells including, but notlimited to adipose, bone marrow, nervous tissue, mammary tissue, livertissue, pancreas, epithelial, respiratory, gonadal and muscle tissue. Inspecific embodiments, the differentiable cells are embryonic stem cells.In other specific embodiments, the differentiable cells are adult stemcells. In still other specific embodiments, the stem cells areplacental- or chorionic-derived stem cells.

Of course, the invention contemplates using differentiable cells fromany animal capable of generating differentiable cells. The animals fromwhich the differentiable cells are harvested may be vertebrate orinvertebrate, mammalian or non-mammalian, human or non-human. Examplesof animal sources include, but are not limited to, primates, rodents,canines, felines, equines, bovines and porcines.

The differentiable cells of the present invention can be derived usingany method known to those of skill in the art. For example, humanpluripotent cells can be produced using de-differentiation and nucleartransfer methods. Additionally, the human ICM/epiblast cell or theprimitive ectoderm cell used in the present invention can be derived invivo or in vitro. Primitive ectodermal cells may be generated inadherent culture or as cell aggregates in suspension culture, asdescribed in WO 99/53021. Furthermore, the human pluripotent cells canbe passaged using any method known to those of skill in the art,including, manual passaging methods, and bulk passaging methods such asenzymatic or non-enzymatic passaging.

In certain embodiment, when ES cells are utilized, the embryonic stemcells have a normal karyotype, while in other embodiments, the embryonicstem cells have an abnormal karyotype. In one embodiment, a majority ofthe embryonic stem cells have a normal karyotype. It is contemplatedthat greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or greaterthan 95% of metaphases examined will display a normal karyotype.

In another embodiment, a majority of the embryonic stem cells have anabnormal karyotype. It is contemplated that greater than 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90% or greater than 95% of metaphases examinedwill display an abnormal karyotype. In certain embodiments, the abnormalkaryotype is evident after the cells have been cultured for greater than5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 passages. In one specificembodiment, the abnormal karyotype comprises a trisomy of at least oneautosomal chromosome, wherein the autosomal chromosome is selected fromthe group consisting of chromosomes 1, 7, 8, 12, 14, and 17. In anotherembodiment, the abnormal karyotype comprises a trisomy of more than oneautosomal chromosome, wherein at least one of the more than oneautosomal chromosomes is selected from the group consisting ofchromosomes 1, 7, 8, 12, 14, and 17. In one embodiment, the autosomalchromosome is chromosome 12 or 17. In another embodiment, the abnormalkaryotype comprises an additional sex chromosome. In one embodiment, thekaryotype comprises two X chromosomes and one Y chromosome. It is alsocontemplated that translocations of chromosomes may occur, and suchtranslocations are encompassed within the term “abnormal karyotype.”Combinations of the foregoing chromosomal abnormalities and otherchromosomal abnormalities are also encompassed by the invention.

The compositions and methods comprise a basal salt nutrient solution. Asused herein, basal salt nutrient solution refers to a mixture of saltsthat provide cells with water and certain bulk inorganic ions essentialfor normal cell metabolism, maintain intra- and extra-cellular osmoticbalance, provide a carbohydrate as an energy source, and provide abuffering system to maintain the medium within the physiological pHrange. Examples of basal salt nutrient solutions include, but are notlimited to, Dulbecco's Modified Eagle's Medium (DMEM), Minimal EssentialMedium (MEM), Basal Medium Eagle (BME), RPM1 1640, Ham's F-10, Ham'sF-12, α-Minimal Essential Medium (aMEM), Glasgow's Minimal EssentialMedium (G-MEM), and Iscove's Modified Dulbecco's Medium, and mixturesthereof. In one particular embodiment, the basal salt nutrient solutionis an approximately 50:50 mixture of DMEM and Ham's F12.

It is contemplated that the composition can further comprise traceelements. Trace elements can be purchased commercially, for example,from Mediatech. Non-limiting examples of trace elements include but arenot limited to compounds comprising, aluminum, chlorine, sulfate, iron,cadmium, cobalt, chromium, germanium, sodium, potassium, calcium,phosphate and magnesium. Specific example of compounds containing traceelements include but are not limited to, AlCl₃, AgNO₃, Ba(C₂H₃O₂)₂,CdCl₂, CdSO₄, CoCl₂, CrCl₃, Cr₂(SO₄)₃, CuSO₄, ferric citrate, GeO₂, KI,KBr, LI, molybdic acid, MnSO₄, MnCl₂, NaF, Na₂SiO₃, NaVO₃, NH₄VO₃,(NH₄)₆MO₇O₂₄, NiSO₄₅ RbCl, selenium, Na₂SeO₃, H₂SeO₃, selenite.2Na,selenomethionone, SnCl₂, ZnSO₄, ZrOCl₂, and mixtures and salts thereof.If selenium, selenite or selenomethionone is present, it is at aconcentration of approximately 0.002 to approximately 0.02 mg/L. Inaddition, hydroxylapatite may also be present.

It is contemplated that amino acids can be added to the defined media.Non-limiting examples of such amino acids are Glycine, L-Alanine,L-Alanyl-L-Glutamine, L-Glutamine/Glutamax, L-Arginine hydrochloride,L-Asparagine-H₂O, L-Aspartic acid, L-Cysteine hydrochloride-H₂O,L-Cystine 2HCl, L-Glutamic Acid, L-Histidine hydrochloride-H₂O,L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine,L-Phenylalanine, L-Proline, L-Hydroxyproline, L-Serine, L-Threonine,L-Tryptophan, L-Tyrosine disodium salt dihydrate, and L-Valine. Incertain embodiments, the amino acid is L-Isoleucine, L-Phenylalanine,L-Proline, L-Hydroxyproline, L-Valine, and mixtures thereof.

It is also contemplated that the defined medium can comprise ascorbicacid. Preferably ascorbic acid is present at an initial concentration ofapproximately 1 mg/L to approximately 1000 mg/L, or from approximately 2mg/L to approximately 500 mg/L, or from approximately 5 mg/L toapproximately 100 mg/L, or from approximately 10 mg/L to approximately100 mg/L or approximately at 50 mg/L.

In addition, the compositions and methods may also comprise othercomponents such as serum albumin, transferrin, L-glutamine, lipids,antibiotics, 13-Mercaptoethanol, vitamins, minerals, ATP and similarcomponents may be present. Examples of vitamins that may be presentinclude, but are not limited to vitamins A, B₁, B₂, B₃, B₅, B₆, B₇, B₉,B₁₂, C, D₁, D₂, D₃, D₄, D₅, E, tocotrienols, K₁ and K₂. One of skill inthe art can determine the optimal concentration of minerals, vitamins,ATP, lipids, essential fatty acids, etc., for use in a given culture.The concentration of supplements may, for example, be from about 0.001μM to about 1 mM or more. Specific examples of concentrations at whichthe supplements may be provided include, but are not limited to about0.005 μM, 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1.0 μM, 2.0 μM, 2.5 μM, 3.0μM 4.0 μM, 5.0 μM, 10 μM, 20 μM, 100 μM, etc. In one specificembodiment, the compositions and methods comprise vitamin B₆ andglutamine. In another specific embodiment, the compositions and methodscomprise vitamin C and an iron supplement. In another specificembodiment, the compositions and methods comprise vitamin K₁ and vitaminA. In another specific embodiment, the compositions and methods comprisevitamin D₃ and ATP. In another specific embodiment, the compositions andmethods comprise vitamin B₁₂ and transferrin. In another specificembodiment, the compositions and methods comprise tocotrienols andβ-Mercaptoethanol. In another specific embodiment, the compositions andmethods comprise glutamine and ATP. In another specific embodiment, thecompositions and methods comprise an omega-3 fatty acid and glutamine.In another specific embodiment, the compositions and methods comprise anomega-6 fatty acid and vitamin B₁. In another specific embodiment, thecompositions and methods comprise α-linolenic acid and B₂.

The compositions of the present invention are essentially serum free. Asused herein, “essentially serum free” refers to the absence of serum inthe solutions of the present invention. Serum is not an essentialingredient to the compositions and methods of the present invention.Thus, the presence of serum in any of the compositions should only beattributable to impurities, e.g., from the starting materials orresidual serum from the primary cell culture. For example, essentiallyserum free medium or environment can contain less than 10, 9, 8, 7, 6,5, 4, 3, 2, or 1% serum wherein the presently improved bioactivemaintenance capacity of the medium or environment is still observed. Ina specific embodiment of the present invention, the essentially serumfree composition does not contain serum or serum replacement, or onlycontains trace amounts of serum or serum replacement from the isolationof components of the serum or serum replacement that are added to thedefined media.

The compositions and methods of the present invention also comprise ameans for stimulating ErbB2 tyrosine kinase activity withindifferentiable cells. In one specific embodiment, the compositions andmethods of the present invention comprise the presence of at least oneErbB3 ligand. Typically, an ErbB3 ligand will bind the ErbB3 receptorand dimerize with the ErbB2 receptor. The ErbB2 receptor is, in turn,generally responsible for intracellular tyrosine kinase activity withinthe differentiable cell.

As used herein, “ErbB3 ligand” refers to a ligand that binds to ErbB3,which in turn dimerizes to ErbB2, thus activating the tyrosine kinaseactivity of the ErbB2 portion of the ErbB2/ErbB3 heterodimeric receptor.Non-limiting examples of ErbB3 ligands include Neuregulin-1; splicevariants and isoforms of Neuregulin-1, including but not limited toHRG-13, HRG-α, Neu Differentiation Factor (NDF), AcetylcholineReceptor-Inducing Activity (ARIA), Glial Growth Factor 2 (GGF2), andSensory And Motor Neuron-Derived Factor (SMDF); Neuregulin-2; splicevariants and isoforms of Neuregulin-2, including but not limited toNRG2-β; Epiregulin; and Biregulin.

In one embodiment, the means for stimulating ErbB2-directed tyrosinekinase activity comprise at least one ErbB3 ligand that is selected fromthe group consisting of Neuregulin-1, Heregulin-β (HRG-β), Heregulin-α(HRG-α), Neu differentiation factor (NDF), acetylcholinereceptor-inducing activity (ARIA), glial growth factor 2 (GGF2),motor-neuron derived factor (SMDF), Neuregulin-2, Neuregulin-2β(NRG2-β), Epiregulin, Biregulin and variants and functional fragmentsthereof. In another specific embodiment, the compositions and methods ofthe present invention comprise more than one means for stimulatingErbB2-directed tyrosine kinase activity, such as, but not limited to,using more than one ErbB3 ligand.

In a more specific embodiment of the compositions and methods of thepresent invention, the ErbB3 ligand is HRG-β or a variant or functionalfragment thereof. In one embodiment, the species from which the cultureadditive protein, polypeptide or variant or functional fragment thereofderives is the same as the species of cells that are cultured. Forexample, if mouse ES cells are cultured, an HRG-β with an amino acidsequence that is identical to the mus musculus HRG-β sequence can beused as an additive in culture and is considered to be “of the samespecies.” In other embodiments, the species from which the biologicaladditive derives is different from the cells being cultured. Forexample, if mouse ES cells are cultured, an HRG-β with an amino acidsequence that is identical to the human HRG-β sequence from can be usedas an additive in culture and is considered to be “of differentspecies.”

As used herein, a “functional fragment” is a fragment or splice variantof a full length polypeptide that exerts a similar physiological orcellular effect as the full length polypeptide. The biological effect ofthe functional fragment need not be identical in scope or strength asthe full-length polypeptide, so long as a similar physiological orcellular effect is seen. For example, a functional fragment of HRG-β candetectably stimulate ErbB2-directed tyrosine kinase.

As used herein, the term “variant” includes chimeric or fusionpolypeptides, homologs, analogs, orthologs, and paralogs. In addition, avariant of a reference protein or polypeptide is a protein orpolypeptide whose amino acid sequence is at least about 80% identical tothe reference protein or polypeptide. In specific embodiments, thevariant is at least about 85%, 90%, 95%, 95%, 97%, 98%, 99% or even 100%identical to the reference protein or polypeptide. As used herein, theterms “correspond(s) to” and “corresponding to,” as they relate tosequence alignment, are intended to mean enumerated positions within thereference protein or polypeptide, e.g., wild-type human or mouseneuregulin-1, and those positions in the modified protein or polypeptidethat align with the positions on the reference protein or polypeptide.Thus, when the amino acid sequence of a subject protein or polypeptideis aligned with the amino acid sequence of a reference protein orpolypeptide, the sequence that “corresponds to” certain enumeratedpositions of the reference protein or polypeptide sequence are thosethat align with these positions of the reference sequence, but are notnecessarily in these exact numerical positions of the referencesequence. Methods for aligning sequences for determining correspondingamino acids between sequences are described below.

A polypeptide having an amino acid sequence at least, for example, about95% “identical” to a reference an amino acid sequence encoding, forexample TGF-β, is understood to mean that the amino acid sequence of thepolypeptide is identical to the reference sequence except that the aminoacid sequence may include up to about five modifications per each 100amino acids of the reference amino acid sequence encoding the referenceTGF-β. In other words, to obtain a peptide having an amino acid sequenceat least about 95% identical to a reference amino acid sequence, up toabout 5% of the amino acid residues of the reference sequence may bedeleted or substituted with another amino acid or a number of aminoacids up to about 5% of the total amino acids in the reference sequencemay be inserted into the reference sequence. These modifications of thereference sequence may occur at the N-terminus or C-terminus positionsof the reference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among amino acids in thereference sequence or in one or more contiguous groups within thereference sequence.

As used herein, “identity” is a measure of the identity of nucleotidesequences or amino acid sequences compared to a reference nucleotide oramino acid sequence. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. (See, e.g.,Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York (1988); Biocomputing: Informatics And Genome Projects,Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey (1994); von Heinje, G., Sequence Analysis In MolecularBiology, Academic Press (1987); and Sequence Analysis Primer, Gribskov,M. and Devereux, J., eds., M Stockton Press, New York (1991)). Whilethere exists several methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H. & Lipton, D., Siam J Applied Math48:1073 (1988)). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego (1994) and Carillo, H. & Lipton, D., Siam J AppliedMath 48:1073 (1988). Computer programs may also contain methods andalgorithms that calculate identity and similarity. Examples of computerprogram methods to determine identity and similarity between twosequences include, but are not limited to, GCG program package(Devereux, et al. 1984 Nucleic Acids Research 12:387), BLASTP, ExPASy,BLASTN, FASTA (Atschul et al. 1990, J Molec Biol 215:403) and FASTDB.Examples of methods to determine identity and similarity are discussedin Michaels & Garian 2000, Current Protocols in Protein Science, Vol 1,John Wiley & Sons, Inc., which is incorporated by reference. In oneembodiment of the present invention, the algorithm used to determineidentity between two or more polypeptides is BLASTP.

In another embodiment of the present invention, the algorithm used todetermine identity between two or more polypeptides is FASTDB, which isbased upon the algorithm of Brutlag et al. (1990, Comp. App. Biosci.6:237-245, incorporated by reference herein). In a FASTDB sequencealignment, the query and subject sequences are amino sequences. Theresult of sequence alignment is in percent identity. Parameters that maybe used in a FASTDB alignment of amino acid sequences to calculatepercent identity include, but are not limited to: Matrix=PAM, k-tuple=2,Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0,Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 orthe length of the subject amino sequence, whichever is shorter.

If the subject sequence is shorter or longer than the query sequencebecause of N-terminus or C-terminus additions or deletions, not becauseof internal additions or deletions, a manual correction can be made,because the FASTDB program does not account for N-terminus andC-terminus truncations or additions of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare N- and C-terminus to the reference sequence that are notmatched/aligned, as a percent of the total bases of the query sequence.The results of the FASTDB sequence alignment determinematching/alignment. The alignment percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thiscorrected score can be used for the purposes of determining howalignments “correspond” to each other, as well as percentage identity.Residues of the query (subject) sequences or the reference sequence thatextend past the N- or C-termini of the reference or subject sequence,respectively, may be considered for the purposes of manually adjustingthe percent identity score. That is, residues that are notmatched/aligned with the N- or C-termini of the comparison sequence maybe counted when manually adjusting the percent identity score oralignment numbering.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue reference sequence to determine percent identity. Thedeletion occurs at the N-terminus of the subject sequence and therefore,the FASTDB alignment does not show a match/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 reference sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected.

The invention also provides chimeric or fusion polypeptides. As usedherein, a “chimeric polypeptide” or “fusion polypeptide” comprises atleast a portion of a member of the reference polypeptide operativelylinked to a second, different polypeptide. The second polypeptide has anamino acid sequence corresponding to a polypeptide which is notsubstantially identical to the reference polypeptide, and which isderived from the same or a different organism. With respect to thefusion polypeptide, the term “operatively linked” is intended toindicate that the reference polypeptide and the second polypeptide arefused to each other so that both sequences fulfill the proposed functionattributed to the sequence used. The second polypeptide can be fused tothe N-terminus or C-terminus of the reference polypeptide. For example,in one embodiment, the fusion polypeptide is a GST-IGF-1 fusionpolypeptide in which an IGF-1 sequence is fused to the C-terminus of theGST sequences. Such fusion polypeptides can facilitate the purificationof recombinant polypeptides. In another embodiment, the fusionpolypeptide can contain a heterologous signal sequence at itsN-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of a polypeptide can be increased throughuse of a heterologous signal sequence.

In addition to fragments and fusion polypeptides, the present inventionincludes homologs and analogs of naturally occurring polypeptides.“Homologs” are defined herein as two nucleic acids or polypeptides thathave similar, or “identical,” nucleotide or amino acid sequences,respectively. Homologs include allelic variants, orthologs, paralogs,agonists, and antagonists as defined hereafter. The term “homolog”further encompasses nucleic acid molecules that differ from a referencenucleotide sequence due to degeneracy of the genetic code and thusencode the same polypeptide as that encoded by the reference nucleotidesequence. As used herein, “naturally occurring” refers to a nucleic oramino acid sequence that occurs in nature.

An agonist of a polypeptide can retain substantially the same, or asubset, of the biological activities of the polypeptide. An antagonistof a polypeptide can inhibit one or more of the activities of thenaturally occurring form of the polypeptide.

In another more specific embodiment of the compositions and methods ofthe present invention, the ErbB3 ligand is HRG-β or a variant or afunctional fragment thereof. Additional, non-limiting examples of ErbB3ligands are disclosed in U.S. Pat. Nos. 6,136,558, 6,387,638, and7,063,961, which are incorporated by reference.

Heregulins are generally classified into two major types, alpha andbeta, based on two variant EGF-like domains that differ in theirC-terminal portions. These EGF-like domains, however, are identical inthe spacing of six cysteine residues contained therein. Based on anamino acid sequence comparison, Holmes et al. found that between thefirst and sixth cysteines in the EGF-like domain, HRGs were 45% similarto heparin-binding EGF-like growth factor (HB-EGF), 35% identical toamphiregulin (AR), 32% identical to TGF-α, and 27% identical to EGF.

The 44 kDa neu differentiation factor (NDF) is the rat equivalent ofhuman HRG. Like the HRG polypeptides, NDF has an immunoglobulin (Ig)homology domain followed by an EGF-like domain and lacks a N-terminalsignal peptide. Presently, there are at least six distinct fibroblasticpro-NDFs, classified as either alpha or beta polypeptides, based on thesequences of the EGF-like domains. Isoforms 1 to 4 are characterized onthe basis of a variable stretch between the EGF-like domain andtransmembrane domain. Thus it appears that different NDF isoforms aregenerated by alternative splicing and may perform distincttissue-specific functions. See European Patent No. 505 148; andInternational Patent Publication Nos. WO 93/22424 and WO 94/28133, whichare incorporated by reference.

In one embodiment of the present invention, the compositions and methodsare free of exogenous insulin and insulin substitutes. The phrase“exogenous insulin or insulin substitutes” is used herein to indicateinsulin or insulin substitutes that is/are not intentionally added tothe compositions or methods of the present invention. Thus, in certainembodiments of the present invention, the methods and compositions arefree of insulin or insulin substitutes that are intentionally supplied.The compositions or methods may, however, not necessarily be free ofendogenous insulin. As used herein, “endogenous insulin” indicates thatthe cultured cells may be producing insulin of their own accord whencultured according to the methods of the present invention. Endogenousinsulin also may be used to indicate residual impurities from theprimary cell culture or impurities from the starting materials. Inspecific examples, the compositions and methods of the present containless than 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or1 μg/mL of insulin.

As used herein, the term “insulin” refers to the protein, or variant orfragment thereof that binds to the insulin receptor in normalphysiological concentrations and can induce signaling through theinsulin receptor. The term “insulin” encompasses a protein having thepolypeptide sequence of native human insulin, or of other mammalianinsulin, or of any homologs or variants to these sequences.Additionally, the term insulin encompasses polypeptide fragments thatare capable of binding to the insulin receptor to induce signalingthrough the insulin receptor. The term “insulin substitute” refers toany zinc containing compound that may be used in place of insulin togive substantially similar results as insulin. Examples of insulinsubstitutes include, but are not limited to zinc chloride, zinc nitrate,zinc bromide, and zinc sulfate.

To be clear, insulin-like growth factors are not insulin substitutes orhomologs of insulin, as contemplated in the present invention.Accordingly, in another specific embodiment, the compositions andmethods of the present invention comprise the use of at least oneinsulin-like growth factor (IGF) or a variant or a functional fragmentthereof. In another embodiment, the compositions and methods of thepresent invention are free of any exogenous insulin-like growth factors(IGFs). In specific embodiments, the compositions and methods of thepresent invention contain less than 200, 150, 100, 75, 50, 25, 20, 15,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng/mL of IGF-1.

As used herein, the term “activator of IGF-1R” refers to mitogens thatplay a pivotal role in regulating cell proliferation, differentiation,and apoptosis. The effects of an activator of IGF-1R are typicallymediated through IGF-1R, although they can be mediated through otherreceptors. The IGF-1R is also involved in cell transformation induced bytumor virus proteins and oncogene products, and the interaction isregulated by a group of specific binding proteins (IGFBPs). In addition,a large group of IGFBP proteases hydrolyze IGFBPs, resulting in therelease of bound IGFs that then resume their ability to interact withIGF-IR. For the purpose of this invention, the ligands, the receptors,the binding proteins, and the proteases are all considered to beactivators of IGF-1R. In one embodiment, the activator of IGF-1R isIGF-1, or IGF-2. In a further embodiment, the activator of IGF-1R is anIGF-1 analog. Non-limiting examples of IGF-1 analogs includeLongR3-IGF1, Des(1-3)IGF-1, [Arg³]IGF-1, [Ala³¹]IFG-1,Des(2,3)[Ala³¹]IGF-1, [Leu²⁴]IGF1, Des(2,3)[Leu²⁴]IGF-1, [Leu⁶⁰]IGF-1,[Ala³¹][Leu⁶⁰]IGF-1, [Leu²⁴][Ala³¹]IGF-1, and combinations thereof. In afurther embodiment, the IFG-1 analog is LongR3-IGF1, which is arecombinant analog of human insulin growth factor-1. It is contemplatedthat LongR3-IGF1 is initially present at a concentration ofapproximately 1 ng/mL to approximately 1000 ng/mL, more preferablyapproximately 5 ng/mL to approximately 500 ng/mL, more preferablyapproximately 50 ng/mL to approximately 500 ng/mL, more preferablyapproximately 100 ng/mL to approximately 300 ng/mL, or at aconcentration of approximately 100 ng/mL.

In certain embodiments, the compositions and methods of the presentinvention comprise transforming growth factor beta (TGF-β) or a TGF-βfamily member or variants or functional fragments thereof. As usedherein, the term “member of the TGF-β family” or the like refers togrowth factors that are generally characterized by one of skill in theart as belonging to the TGF-β family, either due to homology with knownmembers of the TGF-β family, or due to similarity in function with knownmembers of the TGF-β family. In particular embodiments of the invention,if the member of the TGF-β family is present, the TGF-β family member orvariant or functional fragment thereof activates SMAD 2 or 3. In certainembodiments, the member of the TGF-β family is selected from the groupconsisting of Nodal, Activin A, Activin B, TGF-13, bone morphogenicprotein-2 (BMP2) and bone morphogenic protein-4 (BMP4). In oneembodiment, the member of the TGF-β family is Activin A.

It is contemplated that if Nodal is present, it is initially present ata concentration of approximately 0.1 ng/mL to approximately 2000 ng/mL,more preferably approximately 1 ng/mL to approximately 1000 ng/mL, morepreferably approximately 10 ng/mL to approximately 750 ng/mL, or morepreferably approximately 25 ng/mL to approximately 500 ng/mL. It iscontemplated that if used, Activin A is initially present at aconcentration of approximately 0.01 ng/mL to approximately 1000 ng/mL,more preferably approximately 0.1 ng/mL to approximately 100 ng/mL, morepreferably approximately 0.1 ng/mL to approximately 25 ng/mL, or mostpreferably at a concentration of approximately 10 ng/mL. It iscontemplated that if present, TGF-β is initially present at aconcentration of approximately 0.01 ng/mL to approximately 100 ng/mL,more preferably approximately 0.1 ng/mL to approximately 50 ng/mL, ormore preferably approximately 0.1 ng/mL to approximately 20 ng/mL.

In additional embodiments of the present invention, the compositions andmethods of the present invention are free of activators of FGFreceptors. There are currently at least 22 known members of the familyof fibroblast growth factors, with these factors binding to one of atleast one of four FGF receptors. As used herein, the term “activator ofan FGF receptor” refers to growth factors that are generallycharacterized by one of skill in the art as belonging to the FGF family,either due to homology with known members of the FGF family, or due tosimilarity in function with known members of the FGF family. In certainembodiments, the activator of an FGF receptor is an FGF, such as, butnot limited to α-FGF and FGF2. In particular embodiments, thecompositions and methods are free of exogenous FGF2. The phrase“exogenous FGF2” is used herein to indicate fibroblast growth factor 2,i.e., basic FGF that is not intentionally added to the compositions ormethods of the present invention. Thus, in certain embodiments of thepresent invention, the methods and compositions are free ofintentionally supplied FGF2. The compositions or methods may, however,not necessarily be free of endogenous FGF2. As used herein, “endogenousFGF2” indicates that the cultured cells may be producing FGF2 of theirown accord when cultured according to the methods of the presentinvention. “Endogenous FGF2” also may be used to indicate residualimpurities from the primary cell culture or impurities from the startingmaterials. In specific examples, the compositions and methods of thepresent contain less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng/mL ofFGF2.

It is contemplated, however, that the compositions and methods of theinvention can include at least one activator of an FGF receptor,including any of the FGF polypeptides, functional fragments thereof orvariants thereof. It is contemplated that if FGF2 is present, it isinitially present at a concentration of approximately 0.1 ng/mL toapproximately 100 ng/mL, more preferably approximately 0.5 ng/mL toapproximately 50 ng/mL, more preferably approximately 1 ng/mL toapproximately 25 ng/mL, more preferably approximately 1 ng/mL toapproximately 12 ng/mL, or most preferably at a concentration ofapproximately 8 ng/mL. In another specific embodiment, the compositionsand methods of the invention can include at least one activator of anFGF receptor, other than FGF2. For example, the compositions and methodsof the present invention may comprise at least one of FGF-7, FGF-10 orFGF-22 or variants or functional fragments thereof. In specificembodiments, a combination of at least two of FGF-7, FGF-10 and FGF-22,or variants or functional fragments thereof, are present. In anotherembodiment, all three of FGF-7, FGF-10 and FGF-22, or variants orfunctional fragments thereof, are present. It is contemplated that ifany of FGF-7, FGF-10 or FGF-22 or variants or functional fragments arepresent, each is initially present at a concentration of approximately0.1 ng/mL to approximately 100 ng/mL, more specifically fromapproximately 0.5 ng/mL to approximately 50 ng/mL, more specificallyfrom approximately 1 ng/mL to approximately 25 ng/mL, more specificallyfrom approximately 1 ng/mL to approximately 12 ng/mL, or mostspecifically at a concentration of approximately 8 ng/mL.

In additional certain embodiments, the compositions and methods of thepresent invention comprise serum albumin (SA). In specific embodiments,the SA is either bovine SA (BSA) or human SA (HAS). In still morespecific embodiments, the concentration of the SA is more than about0.2%, volume to volume (v/v), but less than about 10% v/v. In even morespecific embodiments, the concentration of SA is more than about 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%,2.2%, 2.4%, 2.6%, 2.8%, 3.0%, 3.2%, 3.4%, 3.6%, 3.8%, 4.0%, 4.2%, 4.4%,4.6%, 4.8%, 5.0%, 5.2%, 5.4%, 5.6%, 5.8%, 6.0%, 6.2%, 6.4%, 6.6%, 6.8%,7.0%, 7.2%, 7.4%, 7.6%, 7.8%, 8.0%, 8.2%, 8.4%, 8.6%, 8.8%, 9.0%, 9.2%,9.4%, 9.6% and 9.8% (v/v).

In additional embodiments, the compositions and methods comprise atleast one insoluble substrate. For example, the differentiable cells maybe placed on a cell culture surface that comprises such compounds as,but is not limited to, polystyrene, polypropylene. The surface may, inturn, be coated with an insoluble substrate. In specific embodiments,the insoluble substrate is selected from the group consisting of acollagen, a fibronectin and fragments or variants thereof. Otherexamples of insoluble substrates include, but are not limited to,fibrin, elastin, fibronectins, laminins and nidogens.

Accordingly, the cell culture environments and methods of the presentinvention comprise plating the cells in an adherent culture. As usedherein, the terms “plated” and “plating” refer to any process thatallows a cell to be grown in adherent culture. As used herein, the term“adherent culture” refers to a cell culture system whereby cells arecultured on a solid surface, which may in turn be coated with aninsoluble substrate that may in turn be coated with another surface coatof a substrate, such as those listed below, or any other chemical orbiological material that allows the cells to proliferate or bestabilized in culture. The cells may or may not tightly adhere to thesolid surface or to the substrate. The substrate for the adherentculture may comprise any one or combination of polyornithine, laminin,poly-lysine, purified collagen, gelatin, fibronectin, tenascin,vitronectin, entactin, heparin sulfate proteoglycans, poly glycolyticacid (PGA), poly lactic acid (PLA), and poly lactic-glycolic acid(PLGA). Furthermore, the substrate for the adherent culture may comprisethe matrix laid down by a feeder layer, or laid down by the pluripotenthuman cell or cell culture. As used herein, the term “extracellularmatrix” encompasses solid substrates such as but not limited to thosedescribed above, as well as the matrix laid down by a feeder cell layeror by the pluripotent human cell or cell culture. In one embodiment, thecells are plated on MATRIGELT™-coated plates. In another embodiment, thecells are plated on fibronectin-coated plates. In certain embodiments,if the cells are plated on fibronectin, the plates are prepared bycoating with 10 μg/mL human plasma fibronectin (Invitrogen, #33016-015),diluted in tissue grade water, for 2-3 hours at room temperature. Inanother embodiment, serum can be placed in the medium for up to 24 hoursto allow cells to plate to the plastic. If using serum to promote theattachment of the cells, the media is then removed and the compositions,which are essentially serum-free, are added to the plated cells.

The compositions and methods of the present invention contemplate thatthe differentiable cells are cultured in conditions that are essentiallyfree of a feeder cell or feeder layer. As used herein, a “feeder cell”is a cell that grows in vitro, that is co-cultured with a target celland stabilizes the target cell in its current state of differentiation.As used herein, a “feeder cell layer” can be used interchangeably withthe term “feeder cell.” As used herein, the term “essentially free of afeeder cell” refers to tissue culture conditions that do not containfeeder cells, or that contain a de minimus number of feeder cells. By“de minimus”, it is meant that number of feeder cells that are carriedover to the instant culture conditions from previous culture conditionswhere the differentiable cells may have been cultured on feeder cells.In one embodiment of the above method, conditioned medium is obtainedfrom a feeder cell that stabilizes the target cell in its current stateof differentiation. In another embodiment, the defined medium is anon-conditioned medium, which is a medium that is not obtained from afeeder cell.

As used herein, the term “stabilize,” when used in reference to thedifferentiation state of a cell or culture of cells, indicates that thecells will continue to proliferate over multiple passages in culture,and preferably indefinitely in culture, where most, if not all, of thecells in the culture are of the same differentiation state. In addition,when the stabilized cells divide, the division typically yield cells ofthe same cell type or yield cells of the same differentiation state. Astabilized cell or cell population in general, does not furtherdifferentiate or de-differentiate if the cell culture conditions are notaltered, and the cells continue to be passaged and are not overgrown. Inone embodiment, the cell that is stabilized is capable of proliferationin the stable state indefinitely, or for at least more than 2 passages.In a more specific embodiment, the cells are stable for more than 3passages, 4 passages, 5 passages, 6 passages, 7 passages, 8 passages, 9passages, more than 10 passages, more than 15 passages, more than 20passages, more than 25 passages, or more than 30 passages. In oneembodiment, the cell is stable for greater than approximately 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, or 11 months of continuous passaging. In anotherembodiment, the cell is stable for greater than approximately 1 year ofcontinuous passaging. In one embodiment, stem cells are maintained inculture in a pluripotent state by routine passage in the defined mediumuntil it is desired that they be differentiated. As used herein, theterm “proliferate” refers to an increase in the number cells in a cellculture.

In certain embodiments, the compositions and methods comprise aninactivator of BMP signaling. As used herein, an “inactivator of BMPsignaling” refers to an agent that antagonizes the activity of one ormore BMP proteins or any of their upstream or downstream signalingcomponents through any of its possible signaling pathways. Thecompound(s) used to inactivate BMP signaling can be any compound knownin the art, or later discovered. Non-limiting examples of inactivatorsof BMP signaling include dominant-negative, truncated BMP receptor,soluble BMP receptors, BMP receptor-Fc chimeras, noggin, follistatin,chordin, gremlin, cerberus/DAN family proteins, ventropin, high doseactivin, and amnionless.

In certain embodiments, the compositions and methods can comprise atleast one hormone, cytokine, adipokine, growth hormone or variant orfunctional fragment thereof. It is currently contemplated that incertain embodiments, the growth hormone present in the defined mediumwill be of the same species as the differentiable cells that arecultured with the defined media. Thus, for example, if a human cell iscultured, the growth hormone is human growth hormone. The use of growthhormone that is from a species different than the cultured cells is alsocontemplated. Preferably the hormone, cytokine, adipokine and/or growthhormone is present at an initial concentration of approximately 0.001ng/mL to approximately 1000 ng/mL, more preferably approximately 0.001ng/mL to approximately 250 ng/mL, or more preferably approximately 0.01ng/mL to approximately 150 ng/mL.

Examples of cytokines and adipokines that may be included in thecompositions and methods of the present invention include, but are notlimited to, the four α-helix bundle family of cytokines, theinterleukin-1 (IL-1) family of cytokines, the IL-17 family of cytokinesand the chemokine family of cytokines Of course, the inventioncontemplates members and subclasses of each of these families ofcytokines, such as, but not limited to, the CC chemokines, the CXCchemokines, the C chemokines and the CX₃C chemokines, interferons,interleukins, lymphotoxins, c-kit ligand, granulocyte-macrophagecolony-stimulating factor (GM-CSF), monocyte-macrophagecolony-stimulating factor (M-CSF), granulocyte colony-stimulating factor(G-CSF), leptin, adiponectin, resistin, plasminogen activatorinhibitor-1 (PAI-1), tumor necrosis factor-alpha (TNFα), tumor necrosisfactor-beta (TNFβ), leukemia inhibitory factor, visfatin, retinolbinding protein 4 (RBP4), erythropoietin (EPO), thrombopoietin (THPO).Of course, one of skill in the art will understand that the inventioncontemplates variants or functional fragments of the above-listedfactors.

The present invention relates to methods of culturing differentiablecells, with the methods comprising plating differentiable cells on acell culture surface, providing a basal salt nutrient solution to thecells and providing a means for stimulating ErbB2-directed tyrosinekinase activity in the cells.

In one embodiment, differentiable cells are contacted with at least oneof the compositions of the invention in the absence of serum or serumreplacement, and in the absence of a feeder cell layer, such that thecells are maintained in an undifferentiated state for at least onemonth. Pluripotency can be determined through characterization of thecells with respect to surface markers, transcriptional markers,karyotype, and ability to differentiate to cells of the three germlayers. These characteristics are well known to those of ordinary skillin the art.

The embodiments of this invention describe various differentiable pPSCincluding human pluripotent stem cells such as hESC including but notlimited to CyT49, CyT203, CyT25, BG01, BG02 and MELT, and inducedpluripotent stem (iPS) cells such as iPSC-482c7 and iPSC-603 (CellularDynamics International, Inc., Madison, Wis.) and iPSC-G4 and iPSC-B7(Shinya Yamanaka, Center for iPS Cell Research, Kyoto University);studies using G4 and B7 are described in detail in U.S. patentapplication Ser. No. 12/765,714, entitled CELL COMPOSITIONS DERIVED FROMDEDIFFERENTIATED REPROGRAMMED CELLS, filed Apr. 22, 2010, which isincorporated by reference herein in its entirety. Certain of these humanpluripotent stem cells are registered with national registries such asthe National Institutes of Health (NIH) and listed in the NIH Human StemRegistry (e.g., CyT49 Registration No. #0041). Information on CyT49 andother available cell lines can also found on the worldwide web atstemcells.nih.gov/research/registry. Still other cell lines, e.g., BG01and BG01v, are sold and distributed to the third parties by WiCell®, anaffiliate of the Wisconsin International Stem Cell (WISC) Bank (Catalogname, BG01) and ATCC (Catalog No. SCRC-2002), respectively. While othercell lines described herein may not be registered or distributed by abiological repository such as WiCell® or ATCC, such cell lines areavailable to the public directly or indirectly from the principleinvestigators, laboratories and/or institutions. Public requests forcell lines and reagents, for example, are customary for those skilled inthe art in the life sciences. Typically, transfer of these cells ormaterials is by way of a standard material transfer agreement betweenthe proprietor of the cell line or material and the recipient. Thesetypes of material transfers occur frequently in a research environment,particularly in the life sciences. In fact, Applicant has transferredcells since the time they were derived and characterized, includingCyT49 (2006), CyT203 (2005), CyT25 (2002), BG01 (2001), BG02 (2001),BG03 (2001) and BG01v (2004) through such agreements with commercial andnon-profit industry partners and collaborators. The date in parenthesisindicates the date when the cell line or material was publicallyavailability.

In August 2006, Klimanskaya et al. demonstrated that hESC can be derivedfrom single blastomeres, hence keeping the embryo intact and not causingtheir destruction. Biopsies were performed from each embryo usingmicromanipulation techniques and nineteen (19) ES-cell-like outgrowthsand two (2) stable hESC lines were obtained. These hESC lines were ableto be maintained in an undifferentiated state for over six (6) months,and showed normal karyotype and expression of markers of pluripotency,including Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, nanog and alkalinephosphatase. These hESC can differentiate and form derivatives of allthree (3) embryonic germ layers both in vitro and form in teratomas invivo. These methods to create new stem cell lines without destruction ofembryos addresses the ethical concerns of using human embryos. SeeKlimanskaya et al. 2006 Nature 444:481-5, Epub 2006 Aug. 23, which isincorporated herein by reference in its entirety.

The present studies used certain CyT or BG hES cell lines, however,subsequent to the initial filing on Jun. 16, 2006 of the provisionalpriority application, U.S. Patent Application No. 60/805,039, otherinvestigators have published reports using the originally describedmethods or variations thereof, using other human pluripotent stem celllines, including but not limited to H7, H9, HUES7, H1, HSF6, chHES-8(eh=China), chHES-20, and chHES-22, H9, BG01, BG02, HUES4, HUES8, HUES9,and HUES 2; other induced pluripotent (iPS) stem cells lines such asDiPS-H1 & DIPS-H2 (T1-diabetes specific iPS cells) and other iPS cellsdescribed in U.S. Patent Publication No. 2010-0272695, entitled CELLCOMPOSITIONS DERIVED FROM DEDIFFERENTIATED REPROGRAMMED CELLS, filedApr. 22, 2010, incorporated by reference herein in its entirety; andhuman pluripotent stem cells such as human parthenogenetic stem cell(hpSC). See e.g., D'Amour et al. 2005, Nature Biotechnology23:1534-1541; Cai et al. 2007, Hepatology, 45:1229-39; King et al. 2008,Regen. Med. 3:175-80; Zhou et al. 2008, Stem Cells & Development17:737-750; Brunner et al. 2009, Genome Res. 19:1044-1056; Maehr et al.2009, PNAS 106:15768-15773; Argawal et al. 2008, Stem Cells26:1117-1127; Bingham et al. 2009, Stem Cells & Devel 18:1-10; Borowiaket al. 2009, Cell Stem Cell 4:348-358; Chen et al. 2009, Nature ChemBiology 5:258:265; Revazova et. al. 2007, Cloning Stem Cells 9:432-449;Turovets et. al. 2011, Differentiation 81:292-8, Epub 2011 Feb. 8, whichare incorporated herein by reference in their entireties. Thus, abundantevidence has been provided by the research community at large toestablish that the methods of the present invention are not limited tothe pluripotent cells described herein.

Tables 1 and 2 are non-exhaustive lists of certain hESC and iPSC,respectively, which are available worldwide for research and/orcommercial purposes, and are suitable for use in the methods andcompositions of the present invention. The information in Tables 1 and 2was derived from the literature and publically available databasesincluding, for example, the National Institutes of Health (NIH) StemCell Registry, the Human Embryonic Stem Cell Registry and theInternational Stem Cell Registry located at the University ofMassachusetts Medical School, Worcester, Mass., USA. These databases areperiodically updated as cell lines become available and registrationobtained.

TABLE 1 Human ES cell lines Institution (Country) Name U.S.A. BresaGen,Inc., Athens, Georgia BG01, BG02, BG03; BG04; BG01v (USA) Invitrogen(USA) BG01v/hOG CyThera, Inc., San Diego, CyT49, CyT203, CyT25California (USA) Geron Corporation, Menlo Park, GE01, GE07, GE09, GE13,GE14, GE91, GE92 (H1, H7, H9, H13, H14, California (USA) H9.1, H9.2)University of California, San UC01, UC06 (HSF-1, HSF-6); UCSFB1, UCSFB2,UCSFB3, UCSFB4, Francisco, California (USA) UCSFB5, UCSFB6, UCSFB7,UCSFB8, UCSFB9 & UCSFB10 Wisconsin Alumni Research WA01, WA07, WA09,WA13, WA14 (H1, H7, H9, H13, H14) Foundation, Madison, Wisconsin (USA)Children's Hospital Corporation CHB-1, CHB-2 CHB-3 CHB-4, CHB-5, CHB-6,CHB-8, CHB-9, CHB- (USA) 10, CHB-11 & CHB-12 The Rockefeller University(USA) RUES1, RUES2 & RUES3 Harvard University (USA) HUES1, HUES2, HUES3,HUES4, HUES5, HUES6, HUES7, HUES8, HUES9, HUES10, HUES11, HUES12,HUES13, HUES14, HUES15, HUES16, HUES17, HUES18, HUES19, HUES20, HUES21,HUES22, HUES23, HUES24, HUES25, HUES26, HUES27; HUES28; HUES48; HUES49;HUES53; HUES55 & HUES 56 Mt Sinai Hosp-Samuel Lunenfeld CA1 & CA2Research Institute (USA) Children's Memorial Hospital CM-1, CM-2, CM-5,CM-6, CM-7, CM-8, CM-11, CM-12, CM-13, CM- (USA) 14, CM-16 TheUniversity of Texas Health CR1 & CR2 Science Center at Houston (USA)California Stem Cell, Inc. (USA) CSC14 University of Connecticut Schoolof CSC14, CT1, CT2, CT3, & CT4 Medicine/Dentistry (USA) The ThirdAffiliated Hospital of FY-3PN; FY-hES-1; FY-hES-3; FY-hES-5; FY-hES-7 &FY-hES-8 Guangzhou Medical College (USA) Advanced Cell Technology, Inc.MA 01; MA 09; MA 42; MA 50; MA135; NED 1; NED 2; NED 3 & NED 4 (USA)Stanford University (USA) MFS5 New York University School of NYUES1;NYUES2; NYUES3; NYUES4; NYUES5; NYUES6 & Medicine (USA) NYUES7Reprogenetics, LLC (USA) RNJ7 University of California, Los UCLA1; UCLA2& UCLA3 Angeles (USA) Eastern Virginia Medical School ES-76; ES-78-1;ES-78-2 (USA) Reproductive Genetics Institute RG-222; RG-230; RG-249;RG-308; RG-313; (USA) RG-148; DYSTROPHIA MYOTONICA 1 (DM1), affected,46,XY; RG-153; DYSTROPHIA MYOTONICA 1 (DM1), affected, 46,XX; RG-170;MUSCULAR DYSTROPHY, BECKER TYPE (BMD), affected, 46,XY; RG-186;HUNTINGTON DISEASE (HD), affected, 46,XX; RG-194; HUNTINGTON DISEASE(HD), affected, 46,XY; RG-233; HEMOGLOBIN BETA LOCUS (HBB), affected(HbS/HbS - sickle cell anemia), 46,XX; RG-245; EMERY-DREIFUSS MUSCULARDYSTROPHY, X-LINKED (EDMD), carrier, 47,XXY; RG-246; EMERY-DREIFUSSMUSCULAR DYSTROPHY, X-LINKED (EDMD), affected, 46,XY; RG-271; TORSIONDYSTONIA 1 (DYT1), AUTOSOMAL DOMINANT, affected (N/GAG del), 46,XY;RG-283; MUSCULAR DYSTROPHY, DUCHENNE TYPE (DMD), affected, 46,XY;RG-288; CYSTIC FIBROSIS (CF), affected (deltaF508/deltaF508), 46,XY;RG-289; CYSTIC FIBROSIS (CF), affected (deltaF508/deltaF508), 46,XX;RG-301; MUSCULAR DYSTROPHY, DUCHENNE TYPE(DMD) affected, 46,XY; RG-302;MUSCULAR DYSTROPHY, DUCHENNE TYPE (DMD), carrier, 46,XX; RG-315;NEUROFIBROMATOSIS, TYPE I (NF1), affected (R19 47X/N), 46,XY; RG-316;TUBEROUS SCLEROSIS, TYPE 1(TSC1), affected (N/IVS7 + 1 G-A); RG-316;TUBEROUS SCLEROSIS, TYPE 1(TSC1), affected (N/IVS7 + 1 G-A); RG-320;TUBEROUS SCLEROSIS, TYPE 1(TSC1), affected (N/IVS7 + 1 G-A); RG-326;POPLITEAL PTERYGIUM SYNDROME (PPS), affected (R84H/N), 46,XY; RG-328;FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY 1A (FSHD), affected, 46,XY;RG-330; FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY 1A (FSHD), affected,46,XY; RG-333; FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY 1A (FSHD),affected, 46,XX; RG-356; HEMOGLOBIN ALPHA LOCUS (HBA), affected(-alpha/--), 46,XX; RG-357; EMERY-DREIFUSS MUSCULAR DYSTROPHY, X-LINKED(EDMD), affected, 46,XY; RG-358; EMERY-DREIFUSS MUSCULAR DYSTROPHY,X-LINKED (EDMD), affected, 46,XY; RG-399; FACIOSCAPULOHUMERAL MUSCULARDYSTROPHY 1A (FSHD), affected, 46,XX; RG-401; FACIOSCAPULOHUMERALMUSCULAR DYSTROPHY 1A (FSHD), affected, 46,XX; RG-402;FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY 1A (FSHD), affected, 46,XX;RG-403; FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY 1A (FSHD), affected;RG-404; SPINAL MUSCULAR ATROPHY, TYPE I (SMA1), affected, 46,XY; RG-406;TORSION DYSTONIA 1, AUTOSOMAL DOMINANT (DYT1), affected (N/GAG del);RG-413; BREAST CANCER, FAMILIAL (BRCA2), affected (N/IVS7 GT del) &MULTIPLE ENDOCRINE NEOPLASIA, TYPE I (MEN1), affected (N/3036 4bp del);RG-414; MULTIPLE ENDOCRINE NEOPLASIA, TYPE I (MEN1), affected (N/30364bp del); RG-415; HUNTINGTON DISEASE (HD), affected; RG-416; CYSTICFIBROSIS (CF), affected (deltaF508/1717-1 G-A); RG-417; CYSTIC FIBROSIS(CF), affected (deltaF508/1717-1 G-A); RG-418; HEMOGLOBIN BETA LOCUS(HBB), affected (cd8 + G/ 619del); RG-420; HEMOGLOBIN BETA LOCUS (HBB),affected (cd8 + G/619del); RG-422; CYSTIC FIBROSIS (CF), affected(N1303K/deltaF508); RG-423; CYSTIC FIBROSIS (CF), carrier (N/deltaF508);RG-424; MULTIPLE ENDOCRINE NEOPLASIA, TYPE 2 (MEN2B), affected(M918T/N); RG-426; PELIZAEUS-MERZBACHER DISEASE (PMLD), affected;RG-428; TUBEROUS SCLEROSIS, TYPE 1 (TSC1), affected (N/IVS7 + 1 G-A)South American Instituto de Biociencias, São Paulo BR-1 (Brazil) MiddleEast Technion-Israel Institute of TE03, TE04, TE06 (I 3, I 4, I 6)Technology, Haifa (Israel) Hadassah University Hospital HAD 1; HAD 2;HAD 3; HAD 4; HAD 5; HAD 6 (Israel) Hebrew University of Jerusalem HEFX1Technion-Israel Institute of I3; I3.2; I3.3; 14; 16; 16.2; J3; J3.2Technology Royan Institute (Iran) ARMD.1.H.iPSC.2; BOM.1.H.iPSC.1;CNS.1.H.iPSC.10; CNS.2.H.iPSC.7; FHC.1.H.iPSC.3; GSD.1.H.iPSC.7;HER.1.H.iPSC.1; LCA.1.H.iPSC.1; LHON.1.H.iPSC.5; R.1.H.iPSC.1;R.1.H.iPSC.4; R.1.H.iPSC.9; Royan H1; Royan H10; Royan H2; Royan H3;Royan H4; Royan H5; Royan H6; Royan H7; Royan H8; Royan H9;RP.1.H.iPSC.2; RP2.H.iPSC.3; TYR.1.H.iPSC.1; USH.1.H.iPSC.6 EuropeCellartis AB, Gotenberg (Sweden) SA001, SA002 (Sahlgrenska 1,Sahlgrenska 2); SA002.2; SA003; AS034.1; AS034.1.1; AS034.2; AS038;AS046; FC018; ASo85; AS094; SA111; SA121; SA142; SA167; SA181; SA191;SA196; SA202; SA203; SA211; SA218; SA240; SA279; SA348; SA352; SA399;SA461; SA502; SA506; SA521; SA540; SA611 Karolinska Institutet (Sweden)HS181; HS207; HS235; HS237; HS293; HS306; HS346; HS351; HS356; HS360;HS361; HS362; HS363; HS364; HS366; HS368; HD380; HS382; HS400; HS401;HS402; HS415; HS420; HS422; HS426; HS429; HS429A; HS429B; HS429C;HS429D; HS475; HS480; HS481; HS539 Göteborg University, GöteborgSA04-SA19 (Sahlgrenska 4-Sahlgrenska 19) (Sweden) Karolinska Institute,Stockholm KA08, KA09, KA40, KA41, KA42, KA43 (hICM8, hICM9, hICM40,(Sweden) hICM41, hICM42, hICM43) Geneva University (Switzerland) CH-ES1University of Basel (Switzerland) CH-ES3; CH-ES3; CH-ES5 Roslin CellsLtd (UK) RC2; RC3; RC4; RC5 University of Newcastle upon Tyne NCL-1;NCL-2; NCL-3; NCL-4; NCL-5; NCL-6; NCL-7; NCL-8; NCL-9 (UK) RoslinInstitute (Edinburgh) & RH1; RH2; RH3; RH4; RH5; RH6; RH7; RH9; GeronCorporation (UK) University of Manchester (UK) Man 2 King's CollegeLondon (UK) KCL-001 (formerly WT3) The University of Sheffield, SHEF-1;SHEF-2; SHEF-3; SHEF-4; SHEF-5; SHEF-6; SHEF-7; SHEF-8 Sheffield (UK)Universities of Edinburgh & Edi-1; Edi-2; Edi-3; Edi-4 Oxford;University of Cambridge (UK) Roslin Cells Ltd, Roslin Institute, RCM-1;RC-1; RC-2; RC-3; RC-4; RC-5; RC-6; RC-7; RC-8; RC-9; RC- Universitiesof Edinburgh & 10 Manchester, Central Manchester & Manchester Children'sUniversity Hospitals NHS Trust (UK) King's College London & Guy'sKCL-003-CF1 (formerly CF1); KCL-005-HD1; KCL008-HD-2; KCL009- HospitalTrust/Charitable trans-1; KCL-001 (WT-3); KCL-001 (WT-4) Foundation ofGuy's & St Thomas (UK) Stem Cell Sciences Ltd, Australia MEL-1; MEL-2;MEL-3; MEL-4 (SCS) & Australian Stem Cell Centre (ASCC) University ofEdinburgh (UK) CB660 Axordia Ltd. (UK) Shef-1; Shef-2; Shef-3; Shef-4;Shef-5; Shef-6; Shef-7 University of Nottingham (UK) Nott-1; Nott-2Centre of Regenerative Medicine in ES-2; ES-3; ES-4; ES-5; ES-6; ES-7;ES-8; ES-9; ES-10; ES-11EM; Barcelona (Spain) cFA404-KiPS4F-1;cFA404-KiPS4F-3; KiPS3F-7; KiPS4F-1; KiPS4F-8 Principe Felipe Centro deVAL-3; VAL-4; VAL-5; VAL-6M; VAL-7; VAL-8; VAL-9; VAL-10B Investigacion(Spain) Université Libre de Bruxelles ERA-1; ERA2; ERA-3; ERAMUC-1;ERAMUC-1 (Belgium) Vrije Universiteit Brussel VUB01; VUB02; VUB06;VUB07; VUB03_DM1; VUB04_CF; (Belgium) VUB05_HD; VUB08_MFS; VUB09_FSHD;VUB10_SCA7; VUB11_FXS; VUB13_FXS; VUB14; VUB19_DM1; VUB20_CMT1A;VUB22_CF; VUB23_OI; VUB24_DM1; VUB26; VUB27; VUB28_HD_MFS CentralManchester and Manchester Man 1; Man 2 Children's University HospitalsNHS (UK) Université Paris-Sud 11 (France) CL01; CL02; CL03; PB04; PB05;PB05-1; PB06; PB06-1; PB07; PB08; PB09; PB10 INSERM (France) OSCAR;STR-I-155-HD; STR-I-171-GLA; STR-I-189-FRAXA; STR-I- 203-CFTR;STR-I-209-MEN2a; STR-I-211-MEN2a; STR-I-221-Sca2; STR-I-229-MTMX;STR-I-231-MTMX; STR-I-233-FRAXA; STR-I-251- CFTR; STR-I-301-MFS;STR-I-305-APC; STR-I-315-CMT1a; STR-I- 347-FRAXA; STR-I-355-APC;STR-I-359-APC Masaryk University (Czech CCTL 6; CCTL 8; CCTL 9; CCTL 10;CCTL 12; CCTL 13; CCRL 14 Republic) Aalborg University (Denmark) CLS1;CLS2; CLS3; CLS4 University of Copenhagen LRB001; LRB002; LRB003;LRB004; LRB005; LRB006; LRB007; (Denmark) LRB008; LRB009; LRB010;LRB011; LRB013; LRB014; LRB016; LRB017; LRB018; University of SouthernDenmark KMEB1; KMEB2; KMEB3; KMEB4; KMEB University of Helsinki(Finland) FES21; FES22; FES29; FES30; FES61; FES75 University of Tampere(Finland) Regea 06/015; Regea 06/040; Regea 07/027; Regea 07/046; Regea08/013; Regea 08/017; Regea 08/023; Regea 08/056 Leiden UniversityMedical Center HESC-NL1; HESC-NL2; HESC-NL3; HESC-NL4 (Netherlands)Russian Academy of Sciences ESM01; ESM02; ESM03; (Russia) InstanbulMemorial Hospital MINE: NS-2; NS-3; NS-4; NS-5; NS-6; NS-7; NS-8; NS-9;NS-10; OZ-1; (Turkey) OZ-2; OZ-3; OZ-4; OZ-5; OZ-6; OZ-7; OZ-8 AustraliaMonash University (Australia) Envy Prince of Wales Hospital, SydneyE1C1; E1C2; E1C3; E1C4; Endeavour 1; Endeavour 2; hES3.1; hES3.2;(Australia) hES3.3 Sydney IVF Limited (Australia) SIVF01; SIVF03;SIVF05; SIVF06; SIVF07; SIVF08; SIVF09; SIVF10; SIVF11; SIVF12; SIVF13Asia Kyoto University (Japan) 201B1; 201B2; 201B3; 201B6; 201B7; 243H1;243H7; 246G1; 246G3; 246G4; 246G5; 246G6; khES-1; khES-2; khES-3;Singapore Stem Cell Consortium ESI-013; ESI-014; ESI-017; ESI-027;ESI-035; ESI-049; ESI-051; ESI- 053 ES Cell International Pte Ld ES01,ES02, ES03, ES04, ES05, ES06 (HES-1, HES-2, HES-3, HES-4, (Singapore)HES-5, HES-6 Maria Biotech Co. Ltd. - Maria MB01, MB02, MB03; MB04;MB05; MB06; MB07; MB08; MB09 Infertility Hospital Medical Institute,Seoul (Korea) MizMedi Hospital - Seoul National MI01 (Miz-hES1);Miz-hES2; Miz-hES3; Miz-hES4; Miz-hES5; Miz- University, Seoul (Korea)hES6; Miz-hES7; Miz-hES8; Miz-hES9; Miz-hES10; Miz-hES11; Miz- hES12;Miz-hES13; Miz-hES14; Miz-hES15; Pochon CHA University College ofCHA-hES3; CHA-hES4 Medicine (Korea) Seoul National University (Korea)SNUhES1; SNUhES2; SNUhES3; SNUhES4; SNUhES11; SNUhES16 National Centrefor Biological NC01, NC02, NC03 (FCNCBS1, FCNCBS2, FCNCBS3); BJN-hem19;Sciences/Tata Institute of BJN-hem20 Fundamental Research, Bangalore(India) Reliance Life Sciences, Mumbai RL05, RL07, RL10, RL13, RL15,RL20, RL21 (RLS ES 05, RLS ES 07, (India) RLS ES 10 National Institutefor Research in KIND-1; KIND-2 Reproductive Health (India) TataInstitute of Fundamental FCNCBS1; FCNCBS2; FCNCBS3 Research (India)Kaohsiung Medical University T1; T2; T3; T4; T5 (Taiwan) Central SouthUniversity (China) chESC-3 (H3); chESC-8; chESC-20; chESC-22; EBNA1 + H9Graduate University of Chinese hPES-1; hPES-2 Academy of Sciences(China) Huazhong University of Science hES-8; hES18 and Technology(China) Peking University Third Hospital B4; B7; PKU1; PKU2 (China)Shanghai Jiao Tong University SHhES1 School of Medicine (China) ShangheiSecond Medical SH1; SH2; SH4; SH7; SH28; SH35; SH35a; SH38; SH39; SH42University (China) Sun Yat-sen University (China) CHES-1; SYSU-1; SYSU-2Sun Yat-sen University Second CHE-1; CHE-2; CHE-3 Affiliated Hospital(China) The Third Affiliated Hospital of FY-hES-5; FY-hES-9; FY-hES-10;;FY-hES-11 Guangzhou Medical College (China)

TABLE 2 Listing of human induced pluripontent stem (hIPS) cell linesInstitution Cell Line University of 1. IPS(FORESKIN)-1 (Normal; 46XY;Yu, J., et al. [Thomson]2007, Science. 2007 Wisconsin - 318: 1917-20.)Madison (USA) 2. IPS(FORESKIN)-2 (Normal; 46XY; Yu, J., et al. supra).3. IPS(FORESKIN)-3 (Normal; 46XY Yu, J. et al. supra). 4.IPS(FORESKIN)-4 (Normal; 46XY; Yu, J. et al. supra). 5. IPS(IMR90)-1(Normal; 46XX; Yu, J. et al. supra). 6. IPS(IMR90)-2 (Normal; 46XX; Yu,J. et al. supra). 7. IPS(IMR90)-3 (Normal; 46XX; Yu, J. et al. supra).8. IPS(IMR90)-4 (Normal; 46XX; Yu, J. et al. supra). 9. IPS-SMA-3.5(Normal; 46XY; Type 1 Spinal Muscular Atrophy; Ebert et al. 2009,Nature. 457: 277-80) 10. IPS-SMA-3.6 (Normal; 46XY; Type 1 SpinalMuscular Atrophy; Ebert et al. 2009, supra) 11. IPS-WT (Normal; 46XX;Type 1 Spinal Muscular Atrophy; Ebert et al. 2009, supra) Universityof 1. IPS-1 (Karumbayaram, S. et al. 2009, Stem Cells 27: 806-811;Lowry, et al.. 2008, California, Los Proc Natl Acad Sci USA. 105:2883-8) Angeles (USA) 2. IPS-2 (Karumbayaram, et al. 2009, supra; Lowryet al. 2008, supra) 3. IPS-5 (Lowry et al. 2008, supra) 4. IPS-7 (Lowryet al. 2008, supra) 5. IPS-18 (Karumbayaram et al. 2009, supra; Lowry etal. 2008, supra) 6. IPS-24 (Lowry et al. 2008, supra) 7. IPS-29 (Lowryet al. 2008, supra) Mt. Sinai Hospital 1. (Woltjen et al. 2009, Nature.458: 766-70) (Samuel Lunenfeld 2. 61 (Woltjen et al. 2009, supra)Research Institute; 3. 66 (Woltjen et al. 2009, supra) USA) 4. 67(Woltjen et al. 2009, supra) 5. HIPSC117 (Kaji et al. 2009, Nature 458:771-5) 6. HIPSC121 (Kaji et al. 2009, supra) 7. HIPSC122 (Kaji et al.2009, supra) Children's Hospital - 1. 551-IPS8 (Park et al. 2008, Nature451: 141-6). Boston (USA) 2. ADA-IPS2 ((ADA-SCID) Adenosine DeaminaseDeficiency-related Severe Combined Immunodeficiency (GGG > AGG, exon 7,ADA gene); Park et al. 2008, Stem Cells Cell 134: 877-86) 3. ADA-IPS3((ADA-SCID) Adenosine Deaminase Deficiency-related Severe CombinedImmunodeficiency (GGG > AGG, exon 7, ADA gene); (Park et al. 2008,supra) 4. BJ1-IPS1 (Park et al. 2008, supra) 5. BMD-IPS1 (Male; (BMD)Becker Muscular Dystrophy (Unidentified mutation in dystrophin); (Parket al. 2008, supra) 6. BMD-IPS4 (Normal; 46XY; (BMD) Becker MuscularDystrophy (Unidentified mutation in dystrophin); (Park et al. 2008,supra) 7. DH1CF16-IPS1 (Normal; 46XY; (Park et al. 2008, supra) 8.DH1CF32-IPS2 (Male; Park et al. 2008, supra) 9. DH1F-IPS3-3(Normal;46XY; Park et al. 2008, supra) 10. DMD-IPS1 ((Normal; 46XY; DMD)Duchenne Muscular Dystrophy (Deletion of exon 45-52, dystrophin gene;Park et al. 2008, supra) 11. DMD-IPS2 (Male; (DMD) Duchenne MuscularDystrophy (Deletion of exon 45-52, dystrophin gene; (Park et al. 2008,supra) 12. DS1-IPS4 (Male; Down syndrome (Trisomy 21); Park et al. 2008,supra) 13. DS2-IPS1 (Male; Down syndrome (Trisomy 21); (Park et al.2008, supra) 14. DS2-IPS10 (Male; Down syndrome (Trisomy 21); Park etal. 2008, supra) 15. GD-IPS1(Male; (GD) Gaucher Disease type III (AAC >AGC, exon 9, G-insertion, nucleotide 84 of cDNA, GBA gene; Park et al.2008, supra) 16. GD-IPS3 (Male; (GD) Gaucher Disease type III (AAC >AGC, exon 9, G-insertion, nucleotide 84 of cDNA, GBA gene; Park et al.2008, supra) 17. HFIB2-IPS2 (Park, I. H., et al. 2008. Generation ofhuman-induced pluripotent stem cells Nat Protoc. 3: 1180-6; Park et al.2008, supra) 18. HFIB2-IPS4 (Park, I. H., et al. 2008. Generation ofhuman-induced pluripotent stem cells Nat Protoc. 3: 1180-6; Park et al.2008, supra) 19. HFIB2-IPS5 (Park, I. H., et al. 2008. Generation ofhuman-induced pluripotent stem cells Nat Protoc. 3: 1180-6; Park et al.2008, supra) 20. JDM-IPS1 (Normal, 46XX; Juvenile diabetes mellitus(multifactorial); Park et al. 2008, supra) 21. JDM-IPS1 (Normal, 46XX;Juvenile diabetes mellitus (multifactorial); Park et al. 2008, supra)22. JDM-IPS2 (Female; Juvenile diabetes mellitus (multifactorial); Parket al. 2008, supra) 23. JDM-IPS3 (Female; Juvenile diabetes mellitus(multifactorial); Park et al. 2008, supra) 24. LNSC-IPS2 (Female;Lesch-Nyhan syndrome (carrier, heterozygosity of HPRT1; Park et al.2008, supra) 25. MRC5-IPS7 (Male; Park et al. 2008, supra) 26.MRC5-IPS12 (Normal; 46XY; Park et al. 2008, supra) 27. MRC5-IPS1 (Male;Park et al. 2008, supra) 28. PD-IPS1 (Male; Parkinson disease(multifactorial); Park et al. 2008, supra) 29. SBDS-IPS1 (Male;Swachman-Bodian-Diamond syndrome (IV2 + 2T > C and IV3 − 1G > A, SBDSgene; Park et al. 2008, supra) 30. SBDS-IPS2 31. SBDS-IPS3 (Normal;46XY; Swachman-Bodian-Diamond syndrome (IV2 + 2T > C and IV3 − 1G > A,SBDS gene; Park et al. 2008, supra) Harvard University 1. A29a (46XX;(ALS) Amyotrophic Lateral Sclerosis (L144F [Leu144 > Phe] (USA) dominantallele of the superoxide dismutase (SOD1) gene; Caucasian; Dimos et al.2008, Science 321: 1218-21) 2. A29b (46XX; (ALS) Amyotrophic LateralSclerosis (L144F [Leu144 > Phe] dominant allele of the superoxidedismutase (SOD1) gene; Caucasian; Dimos, J. T., et al. 2008, supra) 3.A29c (46XX; (ALS) Amyotrophic Lateral Sclerosis (L144F [Leu144 > Phe]dominant allele of the superoxide dismutase (SOD1) gene; Caucasian;Dimos, J. T. et al. 2008, supra) Salk Institute (USA) 1. HAIR-IPS1(Aasen, et al. [Belmonte, J. C.] 2008, Nat Biotechnol. 26: 1276-84) 2.HAIR-IPS2 (Aasen, T., et al. 2008, supra) Royan Institute 1.R.1.H.iPSC.1(OCT4, Sox2, KLF4, c-Myc; Human fibroblasts) (Iran) 2.BOM.1.H.iPSC.1 (OCT4, Sox2, KLF4, c-Myc; Human fibroblasts) 3.FHC.1.H.iPSC.3 (OCT4, Sox2, KLF4, c-Myc; Human fibroblasts) 4.GSD.1.H.iPSC.7 (OCT4, Sox2, KLF4, c-Myc; Human fibroblasts) 5.TYR.1.H.iPSC.1 (OCT4, Sox2, KLF4, c-Myc; Human fibroblasts) 6.HER.1.H.iPSC.1 (OCT4, Sox2, KLF4, c-Myc; Human fibroblasts) 7.R.1.H.iPSC.4 (OCT4, Sox2, KLF4, c-Myc; Human fibroblasts) 8.R.1.H.iPSC.9 (OCT4, Sox2, KLF4, c-Myc; Human fibroblasts) 9.RP2.H.iPSC.3 (OCT4, Sox2, KLF4, c-Myc; iPS cells) 10. LCA.1.H.iPSC.1(OCT4, Sox2, KLF4, c-Myc; iPS cells) 11. USH.1.H.iPSC.6 (OCT4, Sox2,KLF4, c-Myc; Human fibroblasts) 12. RP.1.H.iPSC.2 (OCT4, Sox2, KLF4,c-Myc; Human fibroblasts) 13. ARMD.1.H.iPSC.2 (OCT4, Sox2, KLF4, c-Myc;Human fibroblasts) 14. LHON.1.H.iPSC.5 (OCT4, Sox2, KLF4, c-Myc; iPScells) 15. CNS.1.H.iPSC.10 (OCT4, Sox2, KLF4, c-Myc; iPS cells) 16.CNS.2.H.iPSC.7 (OCT4, Sox2, KLF4, c-Myc; iPS cells) Centre of 1.KiPS4F-1 (OCT4, Sox2, KLF4, c-Myc; human foreskin keratinocytes; 46XY)Regenerative 2. KiPS3F-7 (OCT4, Sox2, KLF4); human foreskinkeratinocytes) Medicine in 3. KiPS4F-8 (OCT4, Sox2, KLF4, c-Myc humanforeskin keratinocytes; 46XY) Barcelona (Spain) 4. cFA404-KiPS4F-1(OCT4, Sox2, KLF4, c-Myc; Epidermal keratinocytes; 46XY) 5.cFA404-KiPS4F-3 (OCT4, Sox2, KLF4, c-Myc; Epidermal keratinocytes; 46XY)Université Paris-Sud 1. PB03 (Oct4, Sox2, Lin28, Nanog; PrimaryAmniocytes; 46XX; Lentivirus) 11 (France) 2. PB04 (Oct4, Sox2, Lin28,Nanog; Primary Amniocytes; Beta-Thalassemia affected; 46XY; Lentivirus)3. PB05-1 (Oct4, Sox2, Lin28, Nanog; Primary Amniocytes;Beta-Thalassemia affected; 46XY; Lentivirus) 4. PB05 (Oct4, Sox2, Lin28,Nanog; Primary Amniocytes; Beta-Thalassemia affected; 46XY; Lentivirus)5. PB06 (Oct4, Sox2, Lin28, Nanog; Primary Amniocytes; Down Syndrome;47XY, +21; Lentivirus) 6. PB06-1 (Oct4, Sox2, Lin28, Nanog; PrimaryAmniocytes; Down Syndrome; 47XY, +21; Lentivirus) 7. PB07 (OCT4, Sox2,KLF4, c-Myc; Primary Amniocytes; 46XY; Retrotivirus) 8. PB08 (OCT4,Sox2, KLF4, c-Myc; Primary Amniocytes; 46XY; Retrotivirus) 9. PB09(Oct4, Sox2, Lin28, Nanog; Primary Amniocytes; 46XY; Lentivirus) 10.PB10 (Oct4, Sox2; Primary Amniocytes46XY, Lentivirus) KyotoUniversity 1. 201B1 (human fibroblast; 46XX) (Japan) 2. 201B2 (humanfibroblast; 46XX) 3. 201B3 (human fibroblast; 46XX) 4. 201B6 (humanfibroblast; 46XX) 5. 201B7 (human fibroblast; 46XX) 6. 243H1 (humanfibroblast) 7. 243H7 (human fibroblast) 8. 246B1 (Normal, 46XX) 9. 246B2(Normal, 46XX) 10. 246B3 (Normal, 46XX) 11. 246B4 (Normal, 46XX) 12.246B5 (Normal, 46XX) 13. 246B6 (Normal, 46XX) 14. 246G1 (humanfibroblast; Takahashi et al. 2007, Cell 131: 861-72) 15. 246G3 (humanfibroblast; Takahashi et al. 2007, supra) 16. 246G4 (human fibroblast;Takahashi et al. 2007, supra) 17. 246G5 (human fibroblast; Takahashi etal. 2007, supra) 18. 246G6 (human fibroblast; Takahashi et al. 2007,supra) 19. 253F1 (Normal, 46XX; Takahashi et al. 2007, supra) 20. 253F2(Normal, 46XX; Takahashi et al. 2007, supra) 21. 253F3 (Normal, 46XX;Takahashi et al. 2007, supra) 22. 253F4 (Normal, 46XX; Takahashi et al.2007, supra) 23. 253F5 (Normal, 46XX; Takahashi et al. 2007, supra)Shanghai Institutes 1. HAFDC-IPS-6 (Li et al. 2009, Hum Mol Genet. 200918: 4340-9) for Biological 2. IPS-S (Liao et al. 2008, Cell Res. 18:600-3) Sciences (China)

With regard to iPSCs (induced pluripotent stem cells), Applicant haspreviously described in detail cell aggregate suspension differentiationin U.S. patent application Ser. No. 12/765,714 (U.S. Patent PublicationNo. 2010-0272695), entitled CELL COMPOSITIONS DERIVED FROMDEDIFFERENTIATED REPROGRAMMED CELLS, filed Apr. 22, 2010, which isincorporated herein by reference in its entirety. Human iPSC aggregationis described in more detail in Example 27. U.S. Patent Publication No.2010-0272695 and Example 27 herein, describes inclusion of at least aRho kinase or ROCK inhibitor in the cell culture medium to enhance,increase, and/or promote growth, survival, proliferation and cell-celladhesion of cells. For example, when employing Y-27632 the concentrationcan range from about 0.01 to about 1000 μM, typically about 0.1 to about100 μM, and frequently about 1.0 to about 50 μM, and most often about 5to 20 μM. When Fasudil/HA1077 is used, it can be used at about two orthree-fold the aforementioned Y-27632 concentration. When H-1152 isused, it can be used at about a fraction, e.g., about 1/10th, 1/20th,1/30th, 1/40th, 1/50th or 1/60th, of the amount of the aforementionedY-27632 concentration. The concentration of ROCK-inhibitor used willdepend, in part, on the bioactivity and potency of the inhibitor and theconditions in which it is used. Further, the time or stage for treatingwith the ROCK inhibitor is particularly not limited provided the desiredeffects such as the enhancing, increasing, and/or promoting growth,survival, proliferation and cell-cell adhesion of cells is achieved.

The cell aggregates described herein can be suspended in anyphysiologically acceptable medium, typically chosen according to thecell type(s) involved. The tissue culture media may comprise, forexample, basic nutrients such as sugars and amino acids, growth factors,antibiotics (to minimize contamination) and the like. In anotherembodiment, the differentiable cells are cultured in suspension, usingthe cell media described herein. The term “suspension” as used in thecontext of cell culturing is used as it is in the art. Namely, cellculture suspensions are cell culture environments where the cells orcell aggregates do not adhere to a surface. One of skill in the art willbe familiar with suspension culture techniques, including, but notlimited to, the use of equipment such as flow hoods, incubators and/orequipment used to keep the cells in constant motion, e.g., rotatorplatforms, shakers, etc, if necessary. As used herein, cells are “inmotion” if they are moving, or if their immediate environment is movingrelative to the cells. If the cells are kept “in motion”, the motionwill, in one embodiment, be a “gentle motion” or “gentle agitation” thatis designed to avoid or prevent exposing the cells to shear stress.

A variety of methods of making cell aggregates are known in the art suchas, for example, the “hanging drop” method wherein cells in an inverteddrop of tissue culture medium sink to the bottom of the drop where theyaggregate; shaking cell suspensions in a laboratory flask; and variousmodifications of these techniques. See, e.g., Timmins et al. 2004,Angiogenesis 7:97-103; Dai et al. 1996, Biotech Bioeng 50:349-56; Fotyet al. 1996, Development 122 :1611-20; Forgacs et al. 2001, J. Biophys.74, 2227-2234; Furukawa et al. 1998, Cell Transplantation 10:441-45;Glicklis et al. 2004. Biotech Bioeng 86:672-80; Carpenedo et al. 2007,Stem Cells 25:2224-34; and Korff et al. 2001, FASEB J. 15:447-57, whichare herein incorporated in their entirety be reference. More recently,cell aggregates have been formed by scraping micropatterned coloniesinto suspension, centrifuging colonies out of microtiter plates and intosuspension or using pipets to dislodge and suspend colonies grown inpatterned microwells (Ungrin et al. 2008 PLoS ONE 3:1-12; Bauwens et al,2008 Stem Cells, Published online Jun. 26, 2008). Although such methodscan be used to produce cell aggregates described herein, the cellaggregates produced herein are optimized for synchronousdirected-differentiation as described in D'Amour et al. 2006, supra,Also, unlike these other methods, the methods for producing the cellaggregates in suspension described herein are amenable to large scalemanufacturing.

In general, the cell medium compositions of the present invention arerefreshed at least once every day, but the medium can be changed moreoften or less often, depending of the specific needs and circumstancesof the suspension culture. In vitro, cells are usually grown in culturemedia in a batch mode (i.e., are batch fed) and exposed to various mediaconditions. As described herein, the cells exist in a dish-culture aseither adherent cultures or as cell aggregates in suspension, andmaintained in contact with a surrounding culture medium; and the wastemedia being replaced periodically. In general, the culture medium may berefreshed about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, or any fraction thereof. Inadditional examples, the medium may be refreshed less often such as, butnot limited to, every 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 orevery 2 or more days, or any time frame in between.

Yet, in another embodiment of the invention, perfusion methods areemployed to prevent degradation of growth factors and other agents whichhave to be replaced frequently; or perfusion as a means to deplete wasteproducts from the culture media over a period of time. For example, U.S.Pat. No. 5,320,963 describes a bioreactor for perfusion culture ofsuspension cells. U.S. Pat. No. 5,605,822 describes a bioreactor system,employing stromal cells to provide growth factors, for growth of HSCcells in culture by perfusion. U.S. Pat. No. 5,646,043 describes growthof HSC cells by continuous and periodic perfusion including mediacompositions for growth of HSC cells. U.S. Pat. No. 5,155,035 describesa bioreactor for suspension culture of cells by fluid media rotation.These references are all incorporated herein in their entireties.

In general, the cells that are cultured in suspension in the mediumcompositions of the present invention are “split” or “passaged” everyweek or so, but the cells can be split more often or less often,depending on the specific needs and circumstances of the suspensionculture. For example, the cells may be split every 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14 or more days, or any time frame in between. Asused herein, the term “split” or “passaged” in the context of cellculture is used as it is in the art. Namely, cell culture splitting, orpassaging, is the collection of cells from a previous culture andsubsequent transfer of a smaller number of collected (harvested) cellsinto a new cell culture vessel. In general, passaging cells allows thecells to continue to grow in a healthy cell culture environment. One ofskill in the art will be familiar with the process and methods of cellculture passaging, which may, but not necessarily, involve the use ofenzymatic or non-enzymatic methods that may be used to disaggregatecells that have clumped together during their growth expansion.

In some instances, a degree of cell death may occur in the cultured(suspended and adherent) cells immediately after passaging. In oneembodiment, the differentiable cells can “recover” from passaging, bydelaying the refreshing of the cell medium for more than 24 hours.Thereafter, the cell medium may be changed more frequently. In anotherembodiment, the cell culture medium can further comprise an inhibitor ofcell death. For example, Wantanabe et al. recently disclosed the use ofa Rho-associated kinase inhibitor, Y27632, to protect human ES cellsafter dissociation. See Wantanabe et al. Nat. Biotechnol., 25:681-6862007, which is incorporated by reference. In additional embodiments, thecell culture medium may comprise caspase inhibitors, growth factors orother trophic factors to prevent or attenuate cell death immediatelyafter passaging. Specific examples of compounds that may be usedinclude, but are not limited to, HA 1077, Dihydrochloride,Hydroxyfasudil, Rho Kinase Inhibitor, Rho-Kinase Inhibitor II, RhoKinase Inhibitor III, Kinase Inhibitor IV and Y27632 all of which arecommercially available. In still another embodiment, the compounds orfactors used to prevent or attenuate cell death during or immediatelyafter cell passaging may be removed from the cell culture medium afterthe cells have recovered from the passaging process. In an additionalembodiment, undifferentiated ES cells aggregate effectively in standardbase media and do not require Y27632 or other interventions to maintainviability during dissociation and aggregation.

In additional embodiments, the compositions and methods of the presentinvention may also comprise the presence or use of surfactants. In oneparticular embodiment, the compositions and methods comprise at leastone surfactant in the context of a suspension culture. Surfactants arewell-known in the art and, generally speaking, are amphiphilic innature. In specific embodiments, the present invention comprises the useof at least one surfactant that is either anionic, cationic, non-ionicor zwitterionic. The concentration of the surfactant used in thecompositions and methods of the present invention is a matter of routinescreening and optimization. For example, Owen et al. reported the use ofsurfactants in cell culture techniques for HeLa cells and human amnioticcells. See Owen et al. J. Cell. Sci., 32:363-376 (1978), which isincorporated by reference. Examples of surfactants that may be usedinclude, but are not limited to, Sodium dodecyl sulfate (SDS), ammoniumlauryl sulfate, and other alkyl sulfate salts, Sodium laureth sulfate(SLES), Alkyl benzene sulfonate, Soaps, or fatty acid salts, Cetyltrimethylammonium bromide (CTAB) (hexadecyl trimethyl ammonium bromide),and other alkyltrimethylammonium salts, Cetylpyridinium chloride (CPC),Polyethoxylated tallow amine (POEA), Benzalkonium chloride (BAC),Benzethonium chloride (BZT), Dodecyl betaine, Dodecyl dimethylamineoxide, Cocamidopropyl betaine, Coco ampho glycinate, Alkyl poly(ethyleneoxide), Copolymers of poly(ethylene oxide) and polypropylene oxide) suchas Pluronic F68, Alkyl polyglucosides, such as, but not limited to,Octyl glucoside, Decyl maltoside, Fatty alcohols, Cetyl alcohol, Oleylalcohol, Cocamide MEA, cocamide DEA and cocamide TEA and/orPolyoxyethylene-sorbitane monolaurate (Tween)

The embodiments described herein provide methods for large-scalemanufacturing of proliferating and/or differentiating hESC bymaintaining a low shear environment thereby maintaining operating celldensity in the system and minimizing fluid shear stresses. Inparticular, the present invention provides methods for maintaining a lowshear environment in a eukaryotic cell manufacturing scale-up system byculturing a cell suspension in a 60 mm dish, 6-well plate, a rotatingbottle, a bioreactor (e.g., spinner flasks), a vessel and the like.Alternatively, continuous perfusion systems for culturing cells requiresagitation or movement in the bioreactor or vessel to provide suspensionof the cells, oxygenation and a supply of fresh nutrients, e.g., forgrowth and/or differentiation. To obtain cell suspension, bioreactorvessels typically use one or more movable mechanical agitation devicesthat are also a potential source of shear stress.

Establishing and maintaining a constant, optimized agitating shear rateis important for maintaining cell growth and viability. For exampleincreased shear rate is deleterious in the following aspects: (1)excessive shear increases energy consumption, (2) excessive shearinterferes with diffusion at the membrane surface, (3) excessive shearcan deprive certain compounds of their bioactivities, and (4) excessiveshear can deform cell membranes beyond the threshold bursting tensionleading to cell lysis. It therefore is desirable to maintain shearwithin an optimal range of 5 to 500 sec⁻¹, depending on the diameter ofthe cell aggregate and the sensitivity of the particular cell line tosingle cell dissociation and shear. Exemplary shear rates produced byconfigurations useful in the methods of the invention are shown inExample 17 for aggregate diameters between 100-200 μm and rotationspeeds between 60-140 rpm for a 6-well dish. These values estimate thetime averaged shear stress that occurs in the bulk fluid duringrotation. However, it is expected that the shear stress at the wall ofthe vessel will be higher due to boundary effects. Using the method ofLey et al. supra, the wall shear stress was calculated for rotationspeeds ranging from 60 rpm to 140 rpm and is shown in Examples 17-19.

Still, other examples of means or devices for generating a gentlyagitated cell suspension exist and are well known to one skilled in theart including impellers, such as propellers, or other mechanical means,bladders, fluid or gas flow-based means, ultrasonic standing wavegenerators, rocking or rotating platforms or combinations thereof whichproduce a cell suspension. In the methods of the invention, a rotatingplatform is an exemplary means for suspending the cells in the mediawhen cells are in 6-well plates, generating a shear rate of less than400 sec⁻¹. Regardless of rotator type or mechanism for generatingagitated mixed fluid suspensions, the estimated time-averaged shear rateand shear stress in the bulk fluid provides a normalizing factor bywhich all fluid mixing devices can be related. While the flow regimesamongst the devices may vary in their profile and extent of laminar orturbulent flow, shear calculations provide a basis for equating flow indevices that produce mixing by different mechanisms. For example, for a125 mL spinner flask with an impeller diameter of 4 cm, a vessel widthof 6.4 cm, an impeller angle of 90 degrees, and an impeller width of 0.1cm, a impeller rotation speed of 135 rpm will generate the sametime-average shear rate and shear stress in the bulk fluid as 6-welldish with 5 mL media rotating at 100 rpm for aggregates of 100 μm indiameter.

The method of the present invention can also be used to maintain a lowshear environment in a manufacturing scale-up system for periods of timeranging from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30 days, to more than 40days, to more than 50 days. An exemplary operating time is at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 23, 24, 25, 26, 27, 28, 29, 30 days to more than 40 days, tomore than 50 days.

It is contemplated that the differentiable cells can be passaged usingenzymatic, non-enzymatic, or manual dissociation methods prior to and/orafter contact with the defined medium of the invention. Non-limitingexamples of enzymatic dissociation methods include the use of proteasessuch as trypsin, collagenase, dispase, and ACCUTASE™. In one embodiment,ACCUTASE™ is used to passage the contacted cells. When enzymaticpassaging methods are used, the resultant culture can comprise a mixtureof singlets, doublets, triplets, and clumps of cells that vary in sizedepending on the enzyme used. A non-limiting example of a non-enzymaticdissociation method is a cell dispersal buffer. Manual passagingtechniques have been well described in the art, such as in Schulz et al.2004 Stem Cells, 22:1218-38. The choice of passaging method isinfluenced by the choice of extracellular matrix, if one is present, andis easily determined by one of ordinary skill in the art.

In one specific embodiment, methods of culturing differentiable cellscomprise providing a dissociation solution to a layer of differentiablecells that are contained in a culture chamber prior to dissociation,where the dissociation breaks apart the layer of cells into singlecells. After dissociation, the single cells are placed into a new tissueculture chamber with a stem cell culture solution, wherein the stem cellculture solution comprises a basal salt nutrient solution and an ErbB3ligand. Once cultured, the single stem cells are placed in conditionsthat permit growth and division of the single cells. In another specificembodiment, the methods of culturing differentiable cells compriseproviding a dissociation solution to an aggregation differentiable cellsthat are contained in a culture chamber prior, where the dissociationbreaks apart the aggregates of cells into single cells or smalleraggregates of cells.

The disaggregation solution used in the methods of the present inventioncan be any disaggregation solution capable of breaking apart ordisaggregating the cells into single cells, without causing extensivetoxicity to the cells. Examples of disaggregation solutions include, butare not limited to, trypsin, ACCUTASE™, 0.25% Trypsin/EDTA, TrypLE, orVERSENE™ (EDTA) and trypsin. The methods of the present invention neednot result in every cell of the confluent layer or suspension beingdisaggregated into single cells, provided that at least a few singlecells are disaggregated and capable of being re-cultured.

Either at the beginning of culture, or after passaging, thedifferentiable cells can be seeded at any density, including a singlecell in a culture chamber. The cell density of the seeded cells may beadjusted depending on a variety of factors, including but not limited tothe use of adherent or suspension cultures, the specific recipe of thecell culture media used, the growth conditions and the contemplated useof the cultured cells. Examples of cell culture densities include, butare not limited to, 0.01×10⁵ cells/mL, 0.05×10⁵ cells/mL, 0.1×10⁵cells/mL, 0.5×10⁵ cells/mL, 1.0×10⁵ cells/mL, 1.2×10⁵ cells/mL, 1.4×10⁵cells/mL, 1.6×10⁵ cells/mL, 1.8×10⁵ cells/mL, 2.0×10⁵ cells/mL, 3.0×10⁵cells/mL, 4.0×10⁵ cells/mL, 5.0×10⁵ cells/mL, 6.0×10⁵ cells/mL, 7.0×10⁵cells/mL, 8.0×10⁵ cells/mL, 9.0×10⁵ cells/mL, or 10.0×10⁵ cells/mL, ormore, e.g., up to 5×10⁷ cells/mL have been cultured with good cellsurvival, or any value in between.

In addition to the above, as used herein, the term “operating celldensity” or “operational cell density” or equivalents thereof refers tothat cell density at which a manufacturing process or system will beoperated to obtain the production of a proliferating or differentiatinghES cell culture. Such cell densities are those at which nutrients suchas vitamins, minerals, amino acids or metabolites, as well asenvironmental conditions such as oxygen tension, that are supplied tothe system are sufficient to maintain cellular viability. Alternatively,such cell densities are those at which waste products can be removedfrom the system at a rate sufficient to maintain cellular viability.Such cell densities can be readily determined by one of ordinary skillin the art.

Operating cell densities that may be maintained are those from at leastabout 0.5×10⁶ cells/mL. In a typical scale-up system operating celldensities may be between about 0.5×10⁶ cells/mL and about 25×10⁶cells/mL. Exemplary densities can be between about 2.5×10⁶ cells/mL,22×10⁶ cells/mL and up to 5×10⁷ cells/mL. In the method of theinvention, cell viability is at least about 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% and up to about 100%. Other scale-upsystem operating cell densities and acceptable cell viability levelswill be recognized by those skilled in the art and can be determined bytechniques well known to those of skill in the art. For example, batch(batch fed), fed-batch and continuous feed configurations, celldensities may be between about 0.5×10⁶ cells/mL and 15×10⁶ cells/mL.

Differentiable cells may also be utilized to screen for molecules orfactors that influence their plasticity or other characteristics. Forexample, differentiable cells could be used to identify agents thatinduce apoptosis, differentiation or proliferation, as well as similareffects in differentiated lineages that have been generated from thedifferentiable cells.

Because the compositions and methods of the present invention allow forsingle cell passaging, differentiable cells have been successfullycultured in high-throughput settings, such as, but not limited to,96-well plates and 384-well plates. FIG. 16 shows the morphology andalkaline phosphatase staining of BG02 cells that were cultured inDC-HAIF in both a 96-well and 384-well plate, using the methodsdescribed herein. Briefly, hESCs cells that were split, using ACCUTASE™,and plated in 96-well and 384-well plates and cultured showed a similarplating efficiency as what is observed using other culture dishes. Inaddition, the cells formed colonies, and were expanded successfully over5 days in the smaller environments. These smaller cultures remainedmorphologically undifferentiated, and stained uniformly positive foralkaline phosphatase, a marker of undifferentiated cells. Furthermore,hESCs could also be grown in 96-well culture devices (not shown) thatprovide real-time measurements of impedance, which can be used tomeasure cell proliferation and viability using the RT-CEST™ methods fromACEA Biosciences, Inc. (www.aceabio.com). Such an approach would enablea label-free identification and quantitation of subtle or immediateeffects on differentiable cells, as well as measurements ofproliferation, apoptosis and changes to morphology, in real time.

The compositions and methods of the invention may contain virtually anycombination of the components set out above or described elsewhereherein, provided the compositions and methods comprise a basal saltnutrient solution and a means for stimulating ErbB2 directed tyrosinekinase activity. As one skilled in the art would recognize, thecomponents of the compositions and methods of the invention will varyaccording to the protocol design. Accordingly, one embodiment of thepresent invention relates to culturing differentiable cells in 96-wellplates and/or 384-well plates. Indeed, using the methods andcompositions of the present invention, the cell culture chamber, i.e.,the culture dish, is no longer limited to specific dimensions. Thus, themethods described herein are in no way limited to specific culturechamber dimensions and/or means and devices to generate hES cells.

The compositions and methods described herein have several usefulfeatures. For example, the compositions and methods described herein areuseful for modeling the early stages of human development. Furthermore,the compositions and methods described herein can also serve fortherapeutic intervention in disease states, such as neurodegenerativedisorders, diabetes mellitus or renal failure, such as by thedevelopment of pure tissue or cell type.

The cell types that differentiate from differentiable cells have severaluses in various fields of research and development including but notlimited to drug discovery, drug development and testing, toxicology,production of cells for therapeutic purposes as well as basic scienceresearch. These cell types express molecules that are of interest in awide range of research fields. These include the molecules known to berequired for the functioning of the various cell types as described instandard reference texts. These molecules include, but are not limitedto, cytokines, growth factors, cytokine receptors, extracellular matrix,transcription factors, secreted polypeptides and other molecules, andgrowth factor receptors.

It is contemplated that the differentiable cells of the invention can bedifferentiated through contact with a cell differentiation environment.As used herein, the term “cell differentiation environment” refers to acell culture condition wherein the differentiable cells are induced todifferentiate, or are induced to become a human cell culture enriched indifferentiated cells. Preferably, the differentiated cell lineageinduced by the growth factor will be homogeneous in nature. The term“homogeneous,” refers to a population that contains more thanapproximately 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the desired cell lineage.

A cell differentiating medium or environment may be utilized topartially, terminally, or reversibly differentiate the differentiablecells of the present invention. In accordance with the invention themedium of the cell differentiation environment may contain a variety ofcomponents including, for example, KODMEM medium (Knockout Dulbecco'sModified Eagle's Medium), DMEM, Ham's F12 medium, FBS (fetal bovineserum), FGF2 (fibroblast growth factor 2), KSR or hLIF (human leukemiainhibitory factor). The cell differentiation environment can alsocontain supplements such as L-Glutamine, NEAA (non-essential aminoacids), P/S (penicillin/streptomycin), N2, B27 and β-mercaptoethanol(β-ME). It is contemplated that additional factors may be added to thecell differentiation environment, including, but not limited to,fibronectin, laminin, heparin, heparin sulfate, retinoic acid, membersof the epidermal growth factor family (EGFs), members of the fibroblastgrowth factor family (FGFs) including FGF2, FGF7, FGF8, and/or FGF10,members of the platelet derived growth factor family (PDGFs),transforming growth factor (TGF)/bone morphogenetic protein (BMP)/growthand differentiation factor (GDF) factor family antagonists including butnot limited to noggin, follistatin, chordin, gremlin, cerberus/DANfamily proteins, ventropin, high dose activin, and amnionless orvariants or functional fragments thereof. TGF/BMP/GDF antagonists couldalso be added in the form of TGF/BMP/GDF receptor-Fc chimeras. Otherfactors that may be added include molecules that can activate orinactivate signaling through Notch receptor family, including but notlimited to proteins of the Delta-like and Jagged families as well asinhibitors of Notch processing or cleavage, or variants or functionalfragments thereof. Other growth factors may include members of theinsulin like growth factor family (IGF), insulin, the wingless related(WNT) factor family, and the hedgehog factor family or variants orfunctional fragments thereof. Additional factors may be added to promotemesendoderm stem/progenitor, endoderm stem/progenitor, mesodermstem/progenitor, or definitive endoderm stem/progenitor proliferationand survival as well as survival and differentiation of derivatives ofthese progenitors.

The compositions described herein are useful for the screening of testcompounds to determine whether a test compound modulates pluripotency,proliferation, and/or differentiation of differentiable cells.Pluripotency, proliferation and/or differentiation of differentiablecells can be readily ascertained by one of ordinary skill in the art.Non-limiting methods include examining cell morphology, the expressionof various markers, teratoma formation, cell counts and measurements ofimpedance.

The progression of the differentiable cells to the desired cell lineage,or its maintenance in an undifferentiated state can be monitored byquantitating expression of marker genes characteristic of the desiredcell lineage as well as the lack of expression of marker genescharacteristic of differentiable cell types. One method of quantitatinggene expression of such marker genes is through the use of quantitativePCR (Q-PCR). Methods of performing Q-PCR are well known in the art.Other methods that are known in the art can also be used to quantitatemarker gene expression. Marker gene expression can be detected by usingantibodies specific for the marker gene of interest.

In certain embodiments, the screening method encompasses methods ofidentifying a compound capable of modulating pluripotency, proliferationand/or differentiation of a differentiable cell, comprising (a)providing a differentiable cell; (b) culturing the cell in a compositioncomprising a basal salt nutrient solution and an ErbB3 ligand, whereinthe composition is essentially serum free; (c) contacting the cell witha test compound; and determining whether an increase or decrease inpluripotency, proliferation and/or differentiation occurs in the cellcontacted with the compound, said increase being an indication that thecompound modulates pluripotency, proliferation and/or differentiation.In certain embodiments, the ErbB3 ligand is HRG-13. In otherembodiments, the ErbB3 ligand can be substituted with a test compound,to determine the effects of the test compound. For example, the effectson pluripotency, proliferation and/or differentiation that occur withthe test compound can be compared to the effects on pluripotency,proliferation and/or differentiation that occurs with the ErbB3 ligandto determine the effects of the test compound on the differentiablecells. It is contemplated that any of the compositions described hereincan be used in the screening methods of the present invention.

In yet another embodiment, the cells can be cultured in the absence ofan ErbB3 ligand (ErbB2-directed tyrosine kinase activity) to determinethe effects of the absence of an ErbB3 ligand (ErbB2-directed tyrosinekinase activity) on the cells.

Using the methods described herein, compositions comprising the desiredcell lineage that are substantially free of other cell types can beproduced. Alternatively, compositions comprising mixtures of thedifferentiable cells and the desired cell lineage can also be produced.

In some embodiments of the present invention, cells of the desired celllineage can be isolated by using an affinity tag that is specific forsuch cells. One example of an affinity tag specific for a target cell isan antibody that is specific to a marker polypeptide that is present onthe cell surface of the target cell but which is not substantiallypresent on other cell types that would be found in a cell cultureproduced by the methods described herein.

The present invention also relates to kits, wherein the kit comprises abasal salt nutrient solution and at least one compound capable ofstimulating ErbB2-directed tyrosine kinase activity. In one embodiment,the kits comprise at least one ErbB3 ligand, as described herein. Inanother embodiment, the kits comprise more than one ErbB3 ligand. Inanother embodiment, the kits comprise at least one of TGF-β or a TGF-βfamily member or a variant or functional fragment thereof as describedherein. In yet another embodiment, the kits comprise more than one ofTGF-β or a TGF-β family member or a variant or functional fragmentthereof. In still another embodiment, the kits comprise at least onefibroblast growth factor or variant or functional fragment thereof. Inanother embodiment, the kits comprise more than one fibroblast growthfactor or variant or functional fragment thereof. In a specificembodiment, the kits comprise at least one of FGF-7, FGF-8, FGF-10,FGF-22 or variants or functional fragments thereof. In anotherembodiment, the kits comprise serum albumin. In still anotherembodiment, the kits comprise serum and/or at least one insolublesubstrate as described herein and/or at least one disaggregationsolution.

The kits of the invention may contain virtually any combination of thecomponents set out above or described elsewhere herein. As one skilledin the art would recognize, the components supplied with kits of theinvention will vary with the intended use for the kits. Thus, kits maybe designed to perform various functions set out in this application andthe components of such kits will vary accordingly.

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in their entirety in order to more fullydescribe the state of the art to which this invention pertains.

EXAMPLES

The human embryonic stem cell line BG01v (BresaGen, Inc., Athens, Ga.)was used in some of the experiments described herein. The BG01v hESCline is a karyotypically variant cell line, which exhibits stablekaryotype containing specific trisomies (karyotype: 49, XXY,+12,+17).Parent cultures were maintained as described previously (Schulz et al.2003, BMC Neurosci., 4:27; Schulz et al. 2004, Stem Cells 22:1218-38;Rosler et al. 2004, Dev. Dynamics, 229:259-274; Brimble et al. 2004 StemCells Dev. 13:585-596). Briefly, the cells were grown in dishes coatedwith MATRIGEL™ or fibronectin, in conditioned media from mouse embryonicfibroblasts (MEFs) (MEF-CM) comprising DMEM:F12 with 20% KSR, 8 ng/mLFGF2, 2 mM L-Glutamine, 1× non-essential amino acids, 0.5 U/mLpenicillin, 0.5 U/mL streptomycin, 0.1 mM β-mercaptoethanol (Sigma, St.Louis, Mo., USA), with collagenase passaging.

The defined culture (DC) media tested herein comprised DMEM/F12, 2 mMGlutamax, 1× non-essential amino acids, 0.5 U/mL penicillin, 0.5 U/mLstreptomycin, 10 μg/mL transferrin (all from Invitrogen, Carlsbad,Calif., USA) 0.1 mM (3-mercaptoethanol (Sigma), 0.2% fatty acid-freeCohn's fraction V BSA (Serologicals), 1× Trace Element mixes A, B and C(Cellgro) and 50 μg/mL Ascorbic Acid (Sigma). Variable levels ofrecombinant growth factors were used, including FGF2 (Sigma),LongR3-IGF1 (JRH Biosciences), Heregulin-13 EGF domain (HRGI3,Peprotech), TGFβ (R&D systems), nodal (R&D systems), LIF (R&D systems),EGF (R&D systems), TGFα (R&D systems), HRGa (R&D systems), BMP4 (R&Dsystems), and Activin A (R&D Systems). LongR3-IGF1 is a modified versionof IGF1 that has reduced affinity for IGF1 binding proteins, some ofwhich are expressed in hESCs. DC-HAIF is the defined culture media asabove, containing 10 ng/mL HRG-13, 10 ng/mL Activin A, 200 ng/mL LR-IGF1and 8 ng/mL FGF2. DC-HAI is defined culture media as above containing 10ng/mL HRG-13, 10 ng/mL Activin A, and 200 ng/mL LR-IGF1. In both DC-HAIFand DC-HAI, the LR-IGF1 component can, of course be replaced with IFG1.

MATRIGEL™ coated dishes were prepared by diluting Growth Factor ReducedBD MATRIGEL™ matrix (BD Biosciences, Franklin Lakes, N.J., USA) to afinal concentration range of about 1:30 to about 1:1000 in coldDMEM/F-12. In one embodiment, the concentration of MATRIGEL™ is about1:200. 1 mL/35 mm dish was used to coat dishes for 1-2 hours at roomtemperature or at least overnight at 4° C. Plates were stored up to oneweek at 4° C. MATRIGEL™ solution was removed immediately before use.

For the tested conditions, parent cultures were plated into 6-welldishes for comparison of multiple conditions. Cultures were typicallyplated directly into the test conditions. The cultures were assessedevery day and graded based on morphological criteria 4 to 5 days afterplating. The grading scale of 1 to 5 involved examining the wholeculture and assessing overall proportion of undifferentiated colonies,their relative size, and proportion of colonies or parts of coloniesexhibiting obvious differentiation. Grade 5 indicates “ideal” cultures,with large undifferentiated colonies and negligible differentiation.Grade 4 indicates a very good culture, but with some obviousdifferentiation. Grade 3 indicates an acceptable culture, but witharound half the colonies exhibiting obvious differentiation. Grade 2cultures are predominantly differentiated, with occasional putativeundifferentiated cells. Grade 1 cultures contain differentiated coloniesor the cultures did not adhere or did not survive. Cultures thatexhibited good expansion of undifferentiated cells were passaged toassess longer-term culture in these conditions.

Example 1 Expression of ErbB1-3, Nrg1 and ADAM19 in BG01v Cells

Real time RT-PCR was used to demonstrate expression of ErbB1-3,Neuregulin and ADAM-19 in BG01v cells (FIG. 1). BG01v cells cultured inDC media as described above, containing 100 ng/mL LongR3-IGF1 (LR-IGF1),8 ng/mL FGF2 and 1 ng/mL Activin A were harvested and RNA was preparedusing the RNeasy mini kit (Qiagen) according to the manufacturer'sinstructions. First strand cDNA was prepared using the iScript kit(Biorad) and real time PCR was carried out using a MJ Research Opticonthermal cycler.

TaqMan assays on demand (Applied Biosystems) for ADAM19 (Hs00224960_m1),EGFR (Hs00193306_m1), ErbB2 (Hs00170433_m1), ErbB3 (Hs00176538_m1), NRG1(Hs00247620_m1), OCT4 (Hs00742896_s1) and control GAPDH were used withTaqMan universal PCR (Applied Biosystems). The real time amplificationplots are shown in FIG. 1, demonstrating expression of these transcriptsin undifferentiated BG01v cells.

Example 2 Inhibition of ErbB2 Slows Proliferation of BG01v Cells

The EGF domain family of ligands bind to the ErbB family of receptortyrosine kinases. To examine the effect of known inhibitors of ErbBtyrosine kinases in hESCs, BG01v cells were plated in 6 well trays onMATRIGEL™ diluted at 1:1000, in defined culture medium (DC) containing100 ng/mL LongR3-IGF1, 8 ng/mL FGF2 and 1 ng/mL Activin A. On the nextday, DMSO (carrier control), 50 nM-20 μM AG1478 (an ErbB1 inhibitor), or100 nM-20 μM AG879 (an ErbB2 inhibitor) was added with fresh medium. Thecells were cultured for an additional 5 days, with daily media changes.The cultures were then fixed and stained for alkaline phosphataseactivity.

Subconfluent colonies of AP+ BG01v cells observed (FIGS. 2A, and B) incontrol and AG1478 cultured cells, indicating that neither DMSO norAG1478 (50 nM-20 μM) had an apparent affect on cell proliferation.AG879, however, substantially inhibited cell growth at 5 μM (FIG. 2C)and caused cell death at 20 μM (not shown). The cultures grown in AG879did not appear to differentiate and appeared to maintain a pluripotentmorphology and alkaline phosphatase activity, indicating that AG879appeared to inhibit proliferation without inducing differentiation,suggesting that BG01v cells are reliant on ErbB2 signaling for cellsurvival. Conversely, BG01v cells grown in similar conditions as abovedo not appear to be reliant on ErbB1 signal for proliferation.

Example 3 BG01v cells are Maintained in Defined Media ContainingHeregulin

Expression of ErbB2 and ErbB3 and the inhibition of proliferation withAG879 suggested that BG01v cells have active endogenous ErbB signalingand that they may also respond to exogenous HRG-13. BG01v cells weregrown in DC medium containing 10 ng/mL HRG-13, 200 ng/mL LongR3-IGF1, 8ng/mL FGF2 and 10 ng/mL Activin A, on MATRIGEL™ diluted 1:1000 (FIGS. 3Aand B). These cells were grown for 4 passages, or >20 days, exhibitedundifferentiated morphology and did not show elevated spontaneousdifferentiation.

Furthermore, BG01v cells were also maintained for 2 passages, or >13days, in DC medium comprising 10 ng/mL HRGβ, 200 ng/mL LongR3-IGF1, and10 ng/mL Activin A. These cultures proliferated normally and exhibitedvery low spontaneous differentiation, demonstrating that BG01v cellscould be maintained in defined conditions with HRGβ in the absence ofFGF2.

Example 4 The Role of ErbB2-Directed Tyrosine Kinase in ES Cells

RT-PCR demonstrated that mESCs express ADAM19, Neuregulin1 (Nrg1), andErbB1-4 (FIG. 4A). In mESCs, ErbB2 and 3 appeared to be expressed athigher levels than ErbB1, with low levels of ErbB4 being detected. Thesedata suggest that endogenous HRG-β could be involved in driving mESCself-renewal.

The expression of the ErbB receptor transcripts in mouse embryonicfibroblasts (MEFs) was also examined (FIG. 4B). MEFs are a heterogenouspopulation of cells derived from E12.5-13.5 viscera that have been usedhistorically to maintain mouse and human EC cells and ES cells.Expression of Nrg1 and Adam19 in this population suggests that the HRG-βectodomain is also present in MEF-conditioned media and may exertsignificant effects upon pluripotency.

AG1478 and AG879 were used to examine the role of HRG/ErbB signaling inmouse ES cells. R1 mouse ES cells were maintained in standard conditionsin DMEM, 10% FBS, 10% KSR, 0.5 U/mL penicillin, 0.5 U/mL streptomycin,1×NEAA, 1 mM sodium pyruvate, 1000 U/mL LIF (ESGRO), 0.1 mM β-ME, andwere passaged with 0.5% trypsin/EDTA. 2×10⁵ cells/well were plated in 6well trays on MATRIGEL™ diluted at 1:1000. The day after plating, DMSO(carrier control), 1-50 μM AG1478, or 1-50 μM AG879 was added with freshmedium. The cells were cultured an additional 8 days, with daily mediachanges. The cultures were then fixed and stained for alkalinephosphatase activity.

DMSO and 1-50 μM AG1478 had no apparent affect on cell proliferation,with subconfluent colonies of alkaline phosphatase positive mESCsobserved (FIGS. 5A-C). However, AG879 substantially inhibited cellgrowth at 50 μM (compare FIGS. 5D and 5F) and may have slowedproliferation at 20 μM (FIG. 5E). mESCs grown in AG879 did not appear todifferentiate and maintained a pluripotent morphology, and alkalinephosphatase activity.

The results indicate that AG879 appeared to inhibit proliferation,without inducing differentiation, of mESCs, suggesting that mESCsrequire ErbB2 signaling for proliferation. Conversely, mESCs do notappear to be reliant on an ErbB1 signal for proliferation. Theconcentration of AG879 required to inhibit proliferation was ˜10× higherfor mESCs than that for BG01v cells grown in defined conditions,indicating that either the serum used in the mESC conditions may haveinterfered with the activity of the drug, that AG879 has a loweraffinity for the mouse ErbB2 tyrosine kinase than for human ErbB2tyrosine kinase, or that ErbB2 may play slightly different roles withthe different species of ES cells.

Another highly selective inhibitor of the ErbB2 tyrosine kinase,tyrphostin AG825 (Murillo, et al. 2001, Cancer Res 61:7408-12), was usedto investigate the role of ErbB2 in human ESCs. AG825 significantlyinhibited proliferation of hESCs growing in conditioned medium (CM)(FIG. 6A). AG825 inhibited proliferation without widespread cell death,and viable hESCs could be maintained for >5 days (not shown). Westernblotting showed that AG825 inhibited autophosphorylation of ErbB2 attyrosine-1248 in starved/heregulin (HRG) pulsed hESCs growing in DC-HAIF(FIG. 6B). Thus, disruption of ErbB2 signaling severely inhibited hESCproliferation. To establish hESCs in defined growth conditions, culturescould be passaged directly from CM conditions into DC-HAIF and exhibitedminimal spontaneous differentiation (FIG. 6C). Colony and cell-countingassays confirmed that LongR3-IGF1 and HRG played the major roles inself-renewal and proliferation in the context of one of the embodimentsof the present invention (FIG. 6D, 6E). Phosphorylation of IGF1R, IR,FGF2a, ErbB2, and ErbB3 was also observed in both steady-state DC-HAIFcultures, and in starved cultures that were pulsed with DC-HAIF (FIG.6F).

Example 5 Culture of Mouse ES Cells in Defined Conditions

To further examine the role of HRG/ErbB2 signaling in mouse ES cells,the proliferation of R1ES cells was examined in DC medium using acombination of growth factors. 1×10⁵ cells/well were plated in 6-welltrays, coated with 0.2% gelatin, in DC containing combinations of 10ng/mL HRG-β, 100 ng/mL LongR3-IGF1, 1 ng/mL Activin A, 1000 U/mL mouseLIF or 10 ng/mL BMP4 (Table 3, below). Proliferation was observed over 8days.

Viable colonies only grew in conditions containing at least LIF/HRG-β orLIF/BMP4 (Table 3). No additional obvious benefit was observed whenLongR3-IGF1 or Activin were added to these combinations. Normalproliferation was observed in a control parental culture, and no viablecolonies were observed in defined media without any growth factors.

TABLE 3 HRG IGF Activin LIF BMP4 Growth + No + + Yes + + No + + +Yes + + + No + + + + Yes + + No + + + Yes + + Yes + + + Yes

A quantitative assay was performed by plating 2×10⁵ cells/well in 6-welltrays on 1:1000 MATRIGEL™, in selected combinations of 10 or 50 ng/mLHRG-13, 10 ng/mL EGF, 1000 U/mL LIF or 10 ng/mL BMP4. The cultures weregrown for 8 days, fixed, and the number of alkaline phosphatase colonieswas counted (FIG. 7A). No colonies were observed in defined conditionswithout growth factors, and <45 colonies were observed with HRG-13,HRG-β/EGF and HRG-β/BMP combinations. While 1358 colonies were observedin LIF alone, 4114 and 3734 colonies were observed in the 10 ng/mLHRG-β/LIF and 50 ng/mL HRG-β/LIF combinations, respectively. Thisindicated that in defined conditions, LIF alone provided a substantialpluripotency signal, and HRG-β exhibited a large synergistic effect withLIF, more than doubling the number of proliferating mESC colonies inthis assay. Low magnification images of this assay also indicate thissynergistic proliferative effect (FIGS. 7B-G).

Example 6 Characterization of Pluripotency of Human Embryonic Stem Cells(hESCs) Maintained in DC-HAIF

Multiple approaches were used to confirm the maintenance of plasticityof hESCs in DC-HAIF. BG02 cells cultured in DC-HAIF for 6 months (25passages) maintained the potential to form complex teratomas (FIG. 8A)and representatives of the three germ layers in vitro (FIG. 8B).Transcriptional analyses were used to compare global expression in hESCscells (Liu et al. 2006, BMC Dev Biol 6:20) maintained in CM and DC-HAIF.Greater than 11,600 transcripts were detected in BG02 cells grown inDC-HAIF for 10 and 32 passages, and BG02 cells grown in CM for 64passages. There were about 10364 transcripts common to all populations(FIG. 8C), including known hESC markers such as CD9, DNMT3, NANOG, OCT4,TERT and UTF1 (not shown). High correlation coefficients were observedin comparisons of CM and DC-HAIF cultures (R²select=0.928), as well asin early and late passage cells (R²select=0.959) (FIG. 8D). Hierarchicalclustering analysis demonstrated that BG02 cells maintained in DC-HAIFgrouped tightly and retained a close similarity to BG02 and BG03 cellsmaintained in CM (FIG. 8E). These data are consistent with previousanalyses showing that undifferentiated hESCs clustered tightly comparedto embryoid bodies or fibroblasts (Liu et al. 2006, BMC Dev Biol 6:20).Thus, cells maintained in the compositions of the present invention areable to maintain key markers of pluripotency. Accordingly, thecompositions of the present invention can be used as a simple medium forsupporting self-renewal of differentiable cells.

Example 7 Maintenance of Human Embryonic Stem Cells (hESCs) on HumanizedExtracellular Matrices (ECMs) in DC-HAIF

To investigate the role of ErbB2 signaling and develop a defined mediafor hESCs, DC-HAIF cultures were initially expanded on culture dishedcoated with growth factor-reduced MATRIGEL™ 1:30, but could also bemaintained successfully long-term on this substrate diluted 1:200 (FIG.9A), or 1:1000. BG02 and CyT49 hESCs could also be maintained for >5passages on tissue culture dishes coated with human serum (FIG. 9B);human fibronectin (FIG. 9C); or VITROGRO™ (FIG. 9D), which is aproprietary humanized ECM.

Example 8 Single Cell Passaging of Human Embryonic Stem Cells (hESCs)

Multiple research groups have demonstrated that certain triplodies,notably of hChrl2 and 17, are accumulated in hESCs under certainsub-optimal culture conditions (Baker et al. 2007, Nat. Biotech.25:207-15). The appearance of triploidies seems to be most directlyrelated to poor cell survival when cultures are split to single cells atpassaging, providing a presumed strong selective growth advantage forcells harboring these aneuploidies. Conversely, hESCs growing in oneembodiment of the present invention, DC-HAIF, maintained high viabilityat plating after being split to single cells (FIG. 10A-D). BG01 and BG02cells maintained a normal karyotype (FIG. 10E) after being passaged withACCUTASE™ for >18 and 19 passages respectively. The maintenance ofnormal karyotype in cells demonstrated that disaggregation of hESCcultures to single cells did not inherently lead to the accumulation ofthese trisomies in hESCs maintained in DC-HAIF. BG01 and BG02 cultureswere also passaged by disaggregation to single cells with multiplepassaging agents (FIG. 11). Cultures were split with ACCUTASE™, 0.25%Trypsin/EDTA, TrypLE, or VERSENE™ (EDTA) for 5 passages and karyotyped.The data demonstrate that culturing and passaging hESCs in thecompositions of the present invention maintained a normal karyotype inat least two human embryonic cell lines, using a variety of celldisaggregation reagents.

Large-scale expansion of undifferentiated hESCs is also possible, usingthe compositions of the present invention. A starting confluent cultureof BG02 cells in a 60 mm plate was expanded in DC-HAIF through 4passages to generate>1.12×10¹⁰ cells in 20 days in a single experiment.The cultures remained undifferentiated, as demonstrated by >85% of thecells in the batch maintaining expression of markers of pluripotencysuch as OCT4, CD9, SSEA-4, TRA-1-81 when examined by flow cytometry(FIG. 12A). Expression of other markers of pluripotency was alsoobserved by RT-PCR analysis, while markers of differentiated lineagesα-fetoprotein, MSX1 and HAND1 were not detected (FIG. 12B). Fluorescencein situ hybridization analysis demonstrated that the cells cultured andpassaged in DC-HAIF maintained expected copy numbers for hChr12 (98%2-copy), hChr17 (98% 2-copy), hChrX (95% 1-copy) and hChrY (98% 1-copy)(FIG. 12C). Karyotyping analysis also demonstrated that a normal euploidchromosome content and banding profile was maintained in these cells.

Example 9 Insulin and IGF1 Exert Different Effects on hESCs When Appliedat Physiological Concentrations

Essentially all of the reported culture conditions for hESCs to dateinclude supraphysiological levels of insulin, which can stimulate bothIR and IGF1R. To distinguish the activities that insulin andinsulin-substitutes exert, compared to IGF1, hESCs are cultured indefined media conditions in physiological levels of these growthfactors. The concentrations of insulin and IGF1 are titrated from about0.2 to about 200 ng/mL and cell proliferation is monitored by countingcells after 5 days. Cultures that expand successfully are seriallypassaged 5 times. Physiological levels of IGF1 support the expansion ofhESC cultures, whereas physiological levels of insulin do not,indicating that the activity of insulin or insulin-substitutes cannotreplace IGF1, and that IGF1 and insulin (or insulin substitutes)represent separate classes of biological activities with regard toaction on hESCs.

Example 10 Methods for Screening the Effects of Supplements

To initially examine the effects of Vitamin B₁₂ and Vitamin B₆ on thegrowth or differentiation hESCs growing at an intermediate density, BG02cells are split using ACCUTASE™ and 1×10⁵ cells/well are plated in6-well trays in defined culture (DC) media. The DC media contains 10ng/mL HRG-β, 200 ng/mL LongR3-IGF1, and 10 ng/mL FGF10. Vitamin B₆ (0.5μM) and/or Vitamin B₁₂ (0.5 μM) are added to experimental wells. Cellnumbers in each condition are counted after 7 days. Cell counting andcolony counting of both experimental and control wells will provideinsight on the effects of Vitamin B₆ and Vitamin B₁₂ on cell growth.

In addition, markers of differentiation, such as OCT4 can be assayed inthe experimental well to determine the effects of the additives andsupplements to the differentiation state of the differentiable cells.

Example 11 Culturing of hESCs in the Absence of FGF2

BG02 cells were maintained long term in DC-HAI, for 20 passages (FIG.13A), and BG01 cells were also serially passaged in DC-HAI, both in theabsence of FGF2. The cultures did not deteriorate or exhibit overtdifferentiation, and exhibited normal expansion of undifferentiatedcolonies throughout the culture period. The maintenance of a normal malekaryotype in a BG02 culture was demonstrated after 6 passages in DC-HAI(FIG. 13B, 20/20 normal metaphase spreads).

Transcriptional analyses were used to compare global expression in hESCscells maintained in DC-HAIF and DC-HAI. Total cellular RNA was isolatedfrom hESCs using Trizol (Invitrogen) and was treated with DNase I(Invitrogen) according to the manufacturer's suggested protocol. Sampleamplification was performed with 100 ng of total RNA using the IlluminaRNA Amplification kit and labeling was achieved by incorporation ofbiotin-16-UTP (Perkin Elmer Life and Analytical Sciences) at a ratio of1:1 with unlabeled UTP. Labeled, amplified material (700 ng per array)was hybridized to Illumina Sentrix Human-6 Expression Beadchipscontaining 47,296 transcript probes according to the manufacturer'sinstructions (Illumina, Inc.). Arrays were scanned with an Illumina BeadArray Reader confocal scanner and primary data processing, backgroundsubtraction, and data analysis were performed using Illumina BeadStudiosoftware according to the manufacturer's instructions. A minimumdetection confidence score of 0.99 (a computed cutoff indicating thetarget sequence signal was distinguishable from the negative controls)was used to discriminate the presence or absence of transcriptexpression. Data analysis was performed using parallel approachesdescribed for other hESC samples (Liu et al. 2006, BMC Dev Biol 6:20).Hierarchical clustering was performed as described previously (Liu etal. 2005, BMC Dev Biol 6:20), and was based on average linkage andEuclidean distances as the similarity metric using differentiallyexpressed genes identified by ANOVA (p<0.05). Detailed descriptions ofthe sensitivity and quality control tests used in array manufacture andalgorithms used in the Bead studio software are available from Illumina,Inc (San Diego, Calif.). The majority of transcripts detected wereexpressed in both DC-HAIF and DC-HAI BG02 cultures, including known hESCmarkers such as CD9, DNMT3, NANOG, OCT4, TERT and UTF1 (not shown). Highcorrelation coefficients were observed in comparisons of DC-HAIF andDC-HAI cultures (R² select=0.961) (FIG. 14). Hierarchical clusteringanalysis demonstrated that BG02 cells maintained in DC-HAI groupedtightly and retained a close similarity to cells maintained in DC-HAIF,as well as BG02 and other hESC lines in multiple culture formats (FIG.15). These data are consistent with previous analyses showing thatundifferentiated hESCs clustered tightly compared to embryoid bodies orfibroblasts (Liu et al. 2006, BMC Dev Biol 6:20).

Furthermore, BG02 cells maintained in DC-HAI differentiated torepresentatives of mesoderm, endoderm and ectoderm in complex teratomasformed in SCID-beige mice (not shown), formally demonstrating themaintenance of pluripotency in cultures grown in the absence ofexogenous FGF2.

To examine if exogenous FGF2 was required in the context of single cellpassaging, BG01 cells were passaged with ACCUTASE™ and grown in definedconditions containing only 10 ng/mL HRG-β and 200 ng/mL LongR3-IGF1(DC-HI). These DC-HI cultures were maintained for 10 passages, and didnot exhibit overt differentiation or a slowing of proliferation.

These studies clearly demonstrated that the provision of exogenous FGF2is not required when hESCs are maintained in defined media minimallycontaining heregulin and IGF1. Furthermore cultures absent FGF2 retainedkey properties of pluripotency, including transcriptional profile anddifferentiation to mesoderm, endoderm and ectoderm in vivo.

Example 12 Suspension Cultures

Starting cultures of BG02 cells were maintained in DC-HAIF medium ondishes coated with 1:200 matrigel, as described herein and were split bypassaging with ACCUTASE™. To initiate suspension culture, BG02 cellswere disaggregated with ACCUTASE™ and placed in low attachment 6-welltrays at a density of 1.6, 3, or 6×10⁵ cells/mL (0.5, 1, or 2×10⁶ cellsin 3 mL volumes) in DC-HAIF medium. The trays were placed on a rotatingplatform at 80-100 rpm in a humidified incubator with 5% CO₂. Underthese conditions hESCs coalesced into small spheres of morphologicallyviable cells within 24 hours.

The medium in the wells was changed on the second day, and every daythereafter. Suspension aggregates continued to proliferate, growinglarger over time without obvious signs of differentiation (FIG. 17).Some of the spheres continued to aggregate over the course of theculture, as some aggregates became much larger than the majority. Inaddition, non-spherical aggregates could be observed in the process ofmerging during the first few days of the culture. To limit thiscontinued aggregation, 38 μg/mL DNaseI was included in some suspensioncultures for the first 24 hours. This approach appeared to be conduciveto the initial aggregation, with relatively larger, but fewer,aggregates formed in the presence of DNaseI. It is not clear, however,if the DNaseI treatment reduced the subsequent merging of spheres andexposure to DNaseI consistently made these aggregates harder to break upwhen splitting.

Suspension cultures were disaggregated with ACCUTASE™ approximatelyevery 7 days and new spheres were established. While the densitiesvaried in different experiments, spheres established within this rangeof densities (1.6−6×10⁵ cells/mL) could be maintained in culture formore than 12 passages, or >80 days, without morphological signs ofdifferentiation. FISH analyses of serially passaged suspension hESCswere also performed to assess the chromosome number for commonaneuplodies. BG02 cells that had been grown in suspension for 6 passagesexhibited normal counts for hChr 12 (96% two copy, n=788), hChr 17 (97%two copy, n=587), hChr X (97% one copy, n=724) and hChr Y (98% one copy,n=689).

Example 13 Expansion of Differentiable Cells in Suspension Culture

Unlike embryoid body culture in the presence of serum or inducers ofdifferentiation, suspension aggregates of hESCs in DC-HAIF did notappear to differentiate. Obvious visceral endoderm was not observed,neither was the formation of structures resembling proamniotic cavities,both classic signs of embryoid body differentiation. To examine the lackof differentiation more closely, cultures were plated back into adherentconditions on MATRIGEL™ diluted 1:200 and cultured in DC-HAIF. Thesecultures were also primarily undifferentiated, and did not exhibitobvious morphological signs of increased differentiation such as thepresence of larger, flattened cells, or structured regions.

Cell counting was used to assess the relative growth rates of cells insuspension compared to adherent culture. In this experiment, an adherentculture of BG02 cells was passaged with ACCUTASE™, and about 1×10⁶ cellswere placed in parallel suspension or adherent culture wells. Individualwells were counted on days 1-6 and plotted on a log scale (FIG. 18).While a higher initial proportion of hESCs were viable after 24 hours inadherent culture (˜90% vs ˜14%), growth rates were comparablethereafter. This indicated that hESCs could proliferate just as rapidlyin suspension culture as in traditional adherent culture. Cell countsperformed during passaging allow one to gauge the amount of expansionpossible in this simple suspension system. In several cultures seededwith 5×10⁵ cells, approximately 10⁷ cells, or more, were generated after7 days. The expansion after 7 days in suspension culture equated toabout a 20-fold or more expansion, with the largest expansion observedbeing ˜24× the input cell number.

Example 14 Characteristics of Differentiable Cells Expanded inSuspension Culture

Quantitative RT-PCR (qPCR) was used to compare gene expression in hESCsgrown in suspension and adherent culture in DC-HAIF. Comparable levelsof OCT4, a marker of pluripotent cells, were observed in both cultureformats, confirming that cultures maintained in suspension wereprimarily undifferentiated. SOX17, a marker of definitive endoderm, wasnot expressed in either population of hESCs. The qPCR analysis alsoexamined the potential of suspension hESCs to differentiate todefinitive endoderm, as aggregates in suspension. Adherent andsuspension hESCs were differentiated using parallel conditions. hESCcultures were treated with RPMI containing 2% BSA, 100 ng/mL Activin A,8 ng/mL FGF2 and 25 ng/mL Wnt3A for 24 hours, followed by 2 days in thesame medium without Wnt3A. The expression of OCT4 was downregulated, andexpression of SOX17 upregulated similarly in both definitive endodermsamples compared to undifferentiated hESCs. This differentiationanalysis confirmed that hESCs cultured in suspension in DC-HAIFmaintained their differentiation potential, as evidenced by the likelyformation of definitive endoderm.

Example 15 Addition of an Apoptosis Inhibitor in Suspension Culture

To attenuate the loss of cells after initial passaging in suspension, aninhibitor of apoptosis was added to the medium. Cells were passaged asin Example 12, except that Y-27632, an inhibitor of p160-Rho-associatedcoiled-coil kinase (ROCK), was added to the medium.

Suspension aggregates of BG02 cells were formed by seeding 2×10⁶ singlecells in 6-well dishes in 3 mL DC-HAIF medium, at 100 rpm on a rotatingplatform in an incubator (Table 4, Experiment A). 10 μM Y27632 ROCKinhibitor was added to test wells for the course of the experiment andthe cultures observed daily and counted after 24 hours (day 1) and after4 or 5 days. As shown in FIG. 20, addition of Y27632 had a profoundeffect on the initial aggregation phase of suspension culture. Comparedto cells aggregated in medium without inhibitor, much larger aggregateswere formed in the presence of Y27632 (FIG. 20). Cell counting confirmedthat more viable cells were present in the presence of inhibitor (Table4, Experiment A). This difference in cell number persisted throughoutthe course of the culture period, with more cells also observed on day4, compared to cultures without inhibitor. As with previous suspensionculture experiments, cells exposed to Y27632 could also be seriallypassaged, and maintained in an undifferentiated state (not shown). Whenthe aggregates were split again, almost twice as many cells wereobserved with Y27632 treatment (Table 4 Experiment A). RT-PCR analysisdemonstrated that BG02 cells grown in suspension culture in the presenceof Y27632 remained undifferentiated (FIG. 21).

As previous experiments had shown that growth rates of cells insuspension and adherent culture were similar after the initial 24 hours,an experiment was performed where Y27632 was removed after this initialperiod (Table 4, Experiment B). Consistent with these previousobservations, Y27632 enhanced initial survival and aggregation of hESCsafter initial passage, but removing the inhibitor after 24 hours did notnegatively impact the number and viability count of cells analyzed onday 5. 1.4×10⁷ (+Y27632) and 1.8×10⁷ (+/−Y27632) viable cells weregenerated when inhibitor was present compared to 3.9×10⁶ cells inuntreated cultures. This analysis confirmed that Y27632 had the largestimpact during the first 24 hours of suspension hESC culture.

Because of the enhanced survival and aggregation observed in thepresence of Y27632, an experiment was performed to examine if it waspossible to reduce the number of cells used to seed suspension cultures(Table 4, Experiment C). Previous experiments had indicated that seedingES cells at a low density of about 5×10⁵ cells per 3 mL DC-HAIF, orless, did not work well. To determine if addition of a ROCK inhibitorwould allow cell seeding at lower densities, a range of cellconcentrations (from about 2×10⁶ total cells down to about 1×10⁵ totalcells was used to seed suspension cultures in 6-well trays cells in 3 mLDC-HAIF. 10 μM Y27632 was added to all conditions, and the cell numberand viability assessed on day 5. Successful aggregation and expansionwas observed even at low seeding densities. An approximately 13 foldexpansion of viable cells was observed even in cultures that were onlyseeded with 1×10⁵ cells Inhibition of ROCK with Y27632 thereforefacilitated initial survival of hESCs at much lower densities in thissuspension system.

TABLE 4 Suspension Cultures with and without an Apoptosis Inhibitor Cellcounts: total Expt. Treatment Seeding (viable, %) p0, day 1 p0, day 4p1, day 4 A HAIF 2 × 10⁶ 1.9 × 10⁶ (3.5 × 10⁵, 19%) 1.8 × 10⁶ (1.3 ×10⁶, 75%) 2.5 × 10⁶ (2.2 × 10⁶, 88%) +Y27632 2 × 10⁶ 1.6 × 10⁶ (1.2 ×10⁶, 74%) 7.8 × 10⁶ (7.1 × 10⁶, 91%) 4.6 × 10⁶ (4.2 × 10⁶, 91%) p0, day5 B HAIF 2 × 10⁶ 2.9 × 10⁶ (5.5 × 10⁵, 26%) 4.8 × 10⁶ (3.9 × 10⁶, 81.3%)+Y27632 2 × 10⁶ 1.9 × 10⁶ (1.4 × 10⁶, 73%) 1.5 × 10⁷ (1.4 × 10⁷, 92%)+/−Y27632 2 × 10⁶ N/A 1.9 × 10⁷ (1.8 × 10⁷, 96%) p0, day 5 C +Y27632 2 ×10⁶ 1.9 × 10⁶ (1.6 × 10⁶, 84%) 1.4 × 10⁷ (1.2 × 10⁷, 90%) 1 × 10⁶ 8.7 ×10⁵ (6.6 × 10⁵, 76%) 8.6 × 10⁶ (7.8 × 10⁶, 91%) 5 × 10⁵ 4.6 × 10⁵ (3.5 ×10⁵, 75%) 5.7 × 10⁶ (5.3 × 10⁶, 93%) 2.5 × 10⁵   2.6 × 10⁵ (2.3 × 10⁵,91%) 2.7 × 10⁶ (2.5 × 10⁶, 91%) 10⁵ 6.8 × 10⁴ (5.4 × 10⁴, 79%) 1.4 × 10⁶(1.3 × 10⁶, 92%) Expt. = Experiment; p0 = passage 0, p1 = passage 1; N/A= not available; Cell counts and percentages are rounded to 1 and 0decimal places, respectively.

Example 16 Suspension Cultures in Various Media

To determine if suspensions of ES cells could be cultured in the absenceof FGF2 and/or Activin A, ES cells were cultured in a variety of media,with and without these factors. Table 5 shows cell counting results fromsuspension cultures and indicate that suspension cultures could besuccessfully expanded in the absence of exogenous FGF2 (HAI conditions),as well as without exogenous FGF2 or Activin A (HI conditions). Theaddition of Y27632 increased the yield of cells generated by day 5 inall conditions. In addition, the cells in each media were successfullypassaged with no morphological signs of differentiation.

TABLE 5 Suspension Cultures in Various Media Cell counts: total (viable,%) Fold Treatment Seeding p0, day 5 Expansion HAIF 2 × 10⁶ 7.7 × 10⁶(6.5 × 10⁶, 83%) 3.25 HAI 2 × 10⁶ 7.0 × 10⁶ (6.3 × 10⁶, 91%) 3.15 HI 2 ×10⁶ 6.4 × 10⁶ (5.3 × 10⁶, 83%) 2.65 HAIF + Y27632 2 × 10⁶ 1.5 × 10⁷ (1.3× 10⁷, 90%) 6.5 HAI + Y27632 2 × 10⁶ 1.5 × 10⁷ (1.3 × 10⁷, 91%) 6.5 HI +Y27632 2 × 10⁶ 1.9 × 10⁷ (9.2 × 10⁶, 49%) 4.6

Example 17 Optimized Shear Rate Results in Increased Survival, UniformDensity and Size of Suspension Cell Aggregates

It is contemplated that any cell line that can be maintained in asuspension cell culture will benefit from and can be utilized inaccordance with the systems, methods and apparatus disclosed herein.Cells include, but are not limited to, mammalian cells, including butnot limited to human cell lines CyT49, CyT203, Cyt25, BG01 and BG02,mouse, dog, and non-human primate stem cell lines, as well as others.

Results provided herein indicate that cell proliferation anddifferentiation can be maintained at control levels or attenuated,depending on the operating parameters of the reactor apparatus,particularly rate of culture flow and provided shear force. The shearforce exerted on cell culture can have significant effects on cellproliferation. A symmetrical system, such as a rotating platformemployed herein, provides a uniform, primarily laminar, shear stressaround the vessel, while an asymmetrical system and mounting, such as astirred-tank bioreactor, has regions of turbulent flow that arecharacterized by locally high shear stress. As such, if the bio-reactoror cell-culture apparatus is not a symmetrical system, the direction ofculture flow affects both the nature and the degree of a shear stressthat results from rotation.

Of course, optimal rotational speeds are culture specific and can varydepending upon cell count in the cell culture, the viscosity of culturemedia, type of media, the robustness of the particular cells insuspension (some cells being able to withstand a higher level of shearforces than others) etc. Optimal rotational speeds are easily determinedfor the particular set of conditions at hand. In particular, rotationalspeeds described and contemplated herein are useful in order to maintainlaminar flow conditions. Therefore, the experiments described hereinwere under conditions where: 1) cell proliferation and differentiationwas maintained at or near control levels; and 2) conditions at whichcell proliferation and differentiation was attenuated. The following isa general method which works well for maintaining hES cell aggregatecultures or differentiated hES cell aggregate cultures. One skilled inthe art can optimize the size and shape of the cell aggregates based onthe description provided herein.

Table 6 below describes shear rate and stress as it relates to thediameter (μm) of the cell aggregates. Human ES cells were aggregated for1, 2, 3 and/or 4 days at various rotation speeds using an orbitalrotator (Barnstead LabLine Multipurpose Rotator): 60 rpm, 80 rpm, 100rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm and 160 rpm. Table 6 alsodemonstrates that the effective shear rate experienced by the cellaggregates depends on the diameter of that cell aggregate.

TABLE 6 Size of cell aggregates is dependent on shear rate and shearstress Shear Aggregate Stress Diameter Rotation Rate Dimensionless(dynes/ Shear Rate (μm) (rpm) Stress cm{circumflex over ( )}2) (1/sec)200 140 0.94 3.16 322.24 120 0.76 2.06 210.12 100 0.59 1.24 126.82 800.43 0.66 67.05 60 0.29 0.30 30.17 175 140 0.72 2.42 246.72 120 0.581.58 160.87 100 0.45 0.95 97.10 80 0.33 0.50 51.33 60 0.22 0.23 23.10150 140 0.53 1.78 181.26 120 0.43 1.16 118.19 100 0.33 0.70 71.34 800.24 0.37 37.71 60 0.16 0.17 16.97 125 140 0.37 1.23 125.88 120 0.300.80 82.08 100 0.23 0.49 49.54 80 0.17 0.26 26.19 60 0.11 0.12 11.79 100140 0.24 0.79 80.56 120 0.19 0.51 52.53 100 0.15 0.31 31.71 80 0.11 0.1616.76 60 0.07 0.07 7.54

To determine how rotation speed controls the diameter of ES aggregates,we generated ES aggregates by rotation at 100 rpm, 120 rpm or 140 rpm.Aggregate diameters were quantitated from 5× phase contrast images takenafter 2 days in rotation culture. For the 100 rpm culture, the averagediameter+/−SD was 198 μm+/−21 μm. For the 120 rpm culture, the averagediameter+/−SD was 225 μm+/−28 μm. For the 140 rpm culture, the averagediameter+/−SD was 85 μm+/−15 μm. Each diameter distribution isstatistically significant (p<0.001) using ANOVA and the Tukey MultipleComparison post-test. As shown in Table 6, the shear rate increasesexponentially from 60 rpm to 140 rpm, e.g., the shear rate for a 100 μmdiameter aggregate was approximately 30 sec-1 at 100 rpm andapproximately 80 sec-1 at 140 rpm, which is about a 3-fold increase.Typically, rotation speeds above 140 rpm resulted in larger, lessuniform hES cell aggregates. Cell aggregate cultures can also becultured initially at reduced rotation speeds, e.g., 60 rpm to 80 rpmfor about 1 day, and then cultured at a higher rotation speed thereafter(e.g., 100 rpm-140 rpm ore more) without any deleterious effects to thesize and or shape of the cell aggregates.

It is important to note, that although the diameters of the cellaggregates varied accordingly with the shear rate, there were noprofound effects in gene expression among the various conditions, i.e.different rotation speeds and/or different size and shaped cellaggregates. That is, the signature markers observed for the pluripotenthESC or the hES-derived cell types (e.g., definitive endoderm, foregutendoderm, PDX1-endoderm, pancreatic endoderm and endocrine cells) wereconsistent with that described in D'Amour et al. supra and relatedapplications incorporated herein by their reference.

To determine the effect of rotation speed, shear rate and shear stresson cell survival or cell viability, it was demonstrated that survivalwas improved by a single day at reduced speeds (e.g., 60 rpm to 80 rpm).For example, cell survival was at least 60% or higher at rotation speedsbetween 60 rpm to 140 rpm. Also, the number of cell aggregates washigher at d1, d2 and d3 in the reduced rotation speed cultures ascompared to higher rotation speeds (e.g., 100 rpm or higher). There wasalso significant disruption and disaggregation when cell aggregates werecultured at the higher rotation speeds (e.g., 140 rpm or higher). Takentogether, these data indicated that cell survival is increased when thecell aggregates were first cultured for at least a single day at reducedrotation speed, however, there was no significant drop in cell survivalwhen rotation speeds were increased to 100 rpm to 140 rpm; although,differentiation at rotation speeds less than 140 rpm is preferred.

Also, culture volume affects shear rate and shear stress which in turn,as discussed above, affects uniformity of size and shape of the cellaggregates. For example, when single cell suspension cultures areinitiated to form cell aggregates in 6 mL as compared to those initiatedin 4 mL resulted in a more uniformly sized and shaped cell aggregates.See FIG. 23, whereby the diameters of the cell aggregates varied fromless than 50 microns to greater than 250 microns when cultured using 4mL, whereas when cultured in 6 mL, the diameters had a tighter range andranged from greater than 50 microns to less 200 microns. Although thedescribed cell aggregates were initiated from single cell suspensioncultures made from adherent hES cell cultures, cell aggregate suspensioncultures initiated from hES-derived adherent plate cultures would beexpected to behave similarly. Thus, the volume of the media is likelyindependent of the stage whereby cell aggregate suspension cultures areinitiated.

Moreover, hES cell aggregates can be cultured in a variety of differentmedia conditions. For example, hES cell aggregate cultures can bemaintained in StemPro® containing media, in DMEM/F12 containing media;or DMEM/F12 containing 20% Knockout serum replacement (KSR, Invitrogen)media; or either StemPro® and DMEM/F12 media further containing 20 ng/mLFGF (R&D Systems) and 20 ng/mL Activin A (R&D Systems); or StemPro® andDMEM/F12 media further containing 10 ng/mL Heregulin B. Alternatively,any of the media mentioned herein and those commercially available canalso be supplemented with xeno-free KSR (Invitrogen). Lastly, cellaggregates were also produced and cultured in any of the above media andfurther not containing exogenous FGF.

Example 18 hES Cell Aggregates in Suspension can Differentiate toEndoderm-Lineage Type Cells

Human embryonic stem (hES) cells were maintained and differentiated invitro to definitive endoderm (stage 1), foregut endoderm and PDX1endoderm substantially as described in D'Amour et al. 2006, supra, andU.S. Patent Publication Numbers 2005/0266554, 2005/0158853,2006/0003313, 2006/0148081, 2007/0122905 and 2007/0259421, which areherein incorporated in their entireties.

Briefly, undifferentiated pluripotent hES adherent (plate) cells weremaintained on mouse embryo fibroblast feeder layers (Millipore, formerlyChemicon or Specialty Media) or on human serum coated 60 mm plates (0.1to 20% final concentration; Valley Biomedical) in DMEM/F12 (Mediatech)supplemented with 20% KnockOut serum replacement (Invitrogen/Gibco), 1mM nonessential amino acids (Invitrogen/Gibco), Glutamax(Invitrogen/Gibco), penicillin/streptomycin (Invitrogen/Gibco), 0.55 mM2-mercaptoethanol (Invitrogen/Gibco) and 4 ng/mL to 20 ng/mL recombinanthuman FGF2 (R&D Systems). Alternatively, the above media can besupplemented with KSR Xeno-free (Gibco) and human serum. Also, humanserum has been added to the culture after the hESC have been seeded onuncoated culture plates. Low dosages of Activin A (2-25 ng/mL, R&DSystems) were added to the growth culture medium to help maintainundifferentiated growth. Adherent pluripotent hESC at day 0 (d0) expresshigh levels of pluripotent protein marker, OCT 4. See FIG. 1, panel A,plate controls at d0.

The cells were either manually or enzymatically passaged againsubstantially as described in D'Amour et al. 2006, supra. The suspensioncultures were dissociated and transferred to a conical tube andcentrifuged at 1000 rpm for about 5 minutes. The supernatant was removedand a standard cell count using a ViCell Cell Analyzer was performed.Typical cell numbers from a 60 mm plate range from 3×10⁶ to 12×10⁶cells, depending on cell line, and the number of days in culture priorto passage. Once the number of cells in the primary cell suspension wasdetermined, the suspension was further diluted with StemPro® or mediacontaining xeno-free KSR as described above to a final volume of 1×10⁶cells/mL. This volume can be increased to >4×10⁶ cells/mL but mayrequire more frequent feeding. ROCK inhibitor Y27632 (Axxora) was addedto the cell suspension to a final concentration of about 1-15 μM,typically 10 μM, and the tube was mixed by gentle inversion. In somecases, Y27632 was not added to the suspension in order to control therate of aggregate formation. The resuspended cells were then distributedequally into each well of a low binding 6-well dish (about 5 mL of cellsuspension per well) and placed on the rotating platform at 100 rpm to140 rpm for about 1-4 days prior to differentiation.

During this culturing period, hES cell aggregates formed and thecultures were fed at least 1-2 times daily by replacing 4 mL of mediawith 4 mL of fresh StemPro® media minus Y27632, or any of the describedmedia supplemented with xeno-free KSR. Media exchanges (“feeding”)should be performed as quickly as possible to disrupt or prevent anyagglomeration and to break the surface tension that may cause aggregatesto float during rotation. Also, to optimize growth and uniformity of thesize and shape of the cell aggregates, the cell aggregates should not beremoved from the rotating platform or apparatus for any long period oftime. Thus, hES cell aggregates can be produced from hES cell adherentcultures which have been well established in the art.

The hES cell aggregates can now be directly differentiated as aggregatesin suspension and substantially as described in D'Amour et al. 2006,supra. Briefly, the StemPro® (minus Y27632) media or any of thedescribed media supplemented with xeno-free KSR was removed from thewells (e.g. aspirated), and the hES cell aggregates washed with 5 mL ofRMPI with no serum (Cat. 15-040-CV; Mediatech), Penicillin/Streptomycin(Invitrogen) and Glutamax (Invitrogen) (also referred to as RMPI,Pen/Strep and Glutamax media), 0% FBS, 1% PenStrep, 1% Glutamax. The6-well dish was then placed back on the rotating platform for 1-2minutes before the wash media was removed. This was repeated at leasttwice or until insulin and/or IGF-I has been sufficiently removed,because although necessary for maintenance of pluripotency and ES selfrenewal, the same factors are detrimental to controlled, synchronous,lineage-directed differentiation. Differentiation to allendoderm-lineages by adding and removing various exogenous mitogens wasperformed at 100 rpm substantially as described by D'Amour et al. 2006,supra, and described in more detail below.

Differentiation to Definitive Endoderm (Stage 1)

Human ES cell aggregates were differentiated in RPMI, 100 ng/mL activinA and varying concentrations of FBS (US Defined FBS, HyClone, catalogueno. SH30070.03), and 25 ng/mL-75 ng/mL Wnt3a for the first day, and inRMPI, Pen/Strep and Glutamax media, further containing 100 ng/mL activinA and varying concentrations of FBS (HyClone) for the second and thirddays (d0 to d2). In most differentiation experiments FBS concentrationswere 0% for the first 24 hours (d1), 0.2% for the second 24 hours (d2),and 0.2% for the third 24 hours (d3), if a three day stage 1 protocolwas used or desired. Preferably a two day stage 1 protocol is performed.

QPCR analysis of hES-derived cell aggregates in suspension culture atthe end of a 2 day stage 1 protocol indicated highly efficient directeddifferentiation of hES aggregates to definitive endoderm as compared tothe adherent plate controls. Cell aggregates were formed at 100 rpm, 120rpm and 140 rpm. In some experiments hES-derived aggregates weretransferred to bioreactors (spinner flasks) prior to differentiation.Adherent hES cell cultures as well as hES cell cultures differentiatedto definitive endoderm cells were used as controls. Increased expressionlevels of SOX17 and FOXA2 were observed in the cell aggregates insuspension and the adherent culture and as compared to undifferentiatedhES cell aggregates and adherent plate controls. See FIG. 22, panelsC(SOX17) & D (FOXA2) at stage 1 (d2). Moreover, expression levels ofSOX7, a gene associated with contaminating extra-embryonic and visceralendoderm, was significantly reduced in the definitive endoderm cellaggregates as compared to the definitive endoderm adherent platecontrols. See FIG. 22, panel L at stage 1 (d2).

Flow cytometric analyses using CXCR4 and HNF3beta (FoxA2) proteinindicated that directed differentiation of ES cell-derived aggregatesresulted in aggregates that were at least 97% CXCR4-positive, at least97% HNF3beta-positive and at least 95% CXCR4/HNF3beta co-positive.

To further evaluate the efficiency of hES cell aggregatedifferentiation, cryosections of ES-derived cell aggregates wereexamined for SOX17 and HNF3beta expression using immunocytochemistry andconfocal microscopy. Image analysis of the stained cryosectionsdemonstrated that greater than ˜90% of all cells at the end of stage 1(definitive endoderm cells) expressed HNF3beta and/or SOX17.

These data all indicate that highly efficient differentiation of EScells as cell aggregates can be achieved, and based on the expressionlevels of signature definitive endoderm markers, the methods forproducing definitive endoderm as described herein are more efficientcompared to differentiation of adherent plate cultures.

Differentiation to PDX1-Negative Foregut Endoderm cells (Stage 2)

Human definitive endoderm cell aggregates from stage 1, were brieflywashed in PBS+/+ and then differentiated in RMPI, Pen/Strep and Glutamaxmedia, further containing 2% FBS, and 25 ng-50 ng/mL KGF (R&D Systems)for another 2 or 3 days. In some experiments 5 μM SB431542 (SigmaAldrich, Inc.) or 2.5 μM TGF-beta Inhibitor IV (Calbiochem) was addedduring the first day of stage 2; and alternatively with RMPI, Pen/Strepand Glutamax media/0.2% FBS/ITS (insulin/transferrin/selenium).

QPCR analysis was performed substantially as discussed above. Increasedexpression levels of HNF1beta and HNF4alpha were observed in the cellaggregate cultures as compared to the adherent plate controls. See FIG.22, panels E (HNF1B) and panel O (HNF4alpha) at stage 2 (d5). Methods ofproducing the specific stage 0, 1, 2 and 5 hES or hES-derived cellaggregates (or “dAggs” for differentiated aggregates) were slightlymodified in panel O. Differentiated cell aggregates in this contextrefers to differentiated hES or hES-derived cell aggregate cultureswhich were initiated from adherent plate control cultures, of thecorresponding stage, from which they were derived. For example, at stage1, differentiated cell aggregates (“dAggs”) suspension cultures werestarted from a stage 0 adherent plate and incubated in any of the mediadescribed herein for about 24 h on a rotating platform at 100 rpm to 140rpm. These differentiated cell aggregates were then furtherdifferentiated to stage 1 definitive endoderm cells with thecorresponding adherent plate controls. FIG. 22, panel 0, shows thatthere is no significant HNF4alpha (HNF4A) expression in either the stage1 differentiated cell aggregates or the adherent plate controls. Incontrast, a similar method was carried out for stage 2 samples andproduced increased expression level of HNF4A. HNF4A expression is alsorobust for stage 5 samples.

Moreover, expression levels of genes associated with extra-embryonicendoderm (SOX7) was significantly reduced in the hES-derived cellaggregate cultures as compared to the plate controls. See FIG. 22, panelL at stage 2 (d5). Thus, demonstrating that directed differentiation ofPDX1-negative foregut endoderm cells by way of cell aggregates insuspension culture removes extra-embryonic endoderm contaminants.

Taken together, these data all indicate that directed differentiation ofhES cell aggregates is highly efficient, and based on the expressionlevels of signature PDX1-negative foregut endoderm markers, the methodsfor producing foregut endoderm cells are improved as compared todifferentiation with adherent plate cultures.

Differentiation to PDXI-Positive Foregut Endoderm Cells (Stage 3)

Foregut endoderm cells from stage 2 were further differentiated in RMPIwith no serum, Glutamax (Invitrogen) and penicillin/streptomycin(Invitrogen), plus 0.5×B27-supplement (Invitrogen/Gibco), and either 1μM to 2 μM retinoic acid (RA, Sigma) and 0.25 nM KAAD-cyclopamine(Toronto Research Chemicals) for 1 to 3 days; or 1 μM to 2 μM retinoicacid, 0.25 nM KAAD-cyclopamine plus 50 ng/mL noggin (R&D systems).Alternatively, 0.2 μM to 0.5 μM RA and 0.25 nM KAAD-cyclopamine wasadded to the media for one day. Still, in some experiments no RA orKAAD-cyclopamine was added to the cell aggregate cultures. Still inother embodiments effective concentrations of 0.1-0.2% BSA were added.

Increased expression levels of PDX1 were observed in hES-derived cellaggregates as compared to the adherent plate controls. See FIG. 22,panel F (PDX1) at stage 3 (d8). Moreover, expression levels of genesassociated with extra-embryonic endoderm (50×7) and visceral endoderm(AFP) was significantly reduced in the hES-derived cell aggregatecultures as compared to the plate controls. See FIG. 22, panel L (SOX7)and panel N (AFP) at stage 3 (d8). Thus, demonstrating that directeddifferentiation to produce PDX1-positive foregut endoderm cells by wayof cell aggregates in suspension culture removes extra-embryonicendoderm contaminants.

Taken together, these data indicate that the directed differentiation ofhES cell aggregates is highly efficient, and based on the expressionlevels of signature PDX1-positive endoderm markers, the methods forproducing PDX1-positive endoderm are improved as compared to theadherent culture controls as compared to the adherent plate controls.

Differentiation to Pancreatic Endoderm or Pancreatic EndocrineProgenitor Cells (Stage 4)

At stage 4, RA is withdrawn from the stage 3 cultures, the cultures werewashed once with DMEM plus B27 (1:100 Gibco), and then the wash isreplaced with either DMEM+1XB27 supplement alone or with anycombinations of or any or all of the following factors: Noggin (50ng/mL), FGF10 (50 ng/mL), KGF (25-50 ng/mL), EGF (25-50 ng/mL), 1-5% FBSfor 4-8 days. In cases where no RA was added, noggin at 30-100 ng/mL(R&D systems) was added to the media for 1-9 days. Further, in someexperiments FGF10 at 25 ng/mL was also added.

Increased expression levels of NKX6.1 and PDX-1 and PTF1A was observedin the ES cell-derived aggregates and the corresponding adherent platecontrols. See FIG. 22, panel F (PDX1), panel G (NKX6.1) and panel P(PTFA1) at stage 4 (d11). In FIG. 22, panel P, the bar chart depictsresults from methods for determining whether hES and/or hES-derived cellaggregates in suspension were affected by the number of cells in anadherent plate culture from which they were derived. Although Panel Ponly shows results for 1×10⁷ cells, cell-aggregate suspension cultureswere started from various seed counts, e.g. 1×10⁶ to 2×10⁷ cells. Allwere substantially similar and produced cell aggregate cultures whichhad good viability and little cell death. For example, at stage 4,differentiated cell aggregate suspension cultures (“dAggs”) were startedfrom a d5 (stage 2) adherent plate, and again incubated in any of themedia described herein for about 24 h on a rotating platform at 100 rpmto 140 rpm. These differentiated cell aggregates were then furtherdifferentiated to stage 4 pancreatic endoderm type cells expressingPTF1A (panel P). As compared to the corresponding stage 4 adherent platecontrols, there was increased expression of PTF1A.

Moreover, expression levels of AFP were significantly reduced in thehES-derived cell aggregates as compared to the adherent plate controls.See FIG. 22, panel N at stage 4 (d11). Thus, demonstrating that directeddifferentiation to produce PDX1-positive pancreatic endoderm cells byway of cell aggregates in suspension culture removes visceral endodermcontaminants.

Flow cytometric analyses using NKX6.1, HNF3beta and Chromogranin (CHG)protein indicated that directed differentiation of hES-derived cellaggregates resulted in cell aggregates that were at least 53%CHG-positive, at least 40% NKX6.1 and CHG co-positive, and small amountof HNF3beta and other types of cells.

Cryosections of hES-derived aggregates were examined for NKX6.1, PDX1and NKX2.2 expression using immunocytochemistry and confocal microscopyat the end of stage 4. Image analysis indicated highly efficientdifferentiation of aggregated cells to pancreatic endoderm (orPDX1-positive pancreatic endoderm), with nearly all cells expressingPDX1 and a large populations of cells expressing NKX6.1 (approximately40% of cells) and/or NKX2.2 (approximately 40% of cells).

Differentiation to Hormone Expressing Endocrine Cells (Stage 5)

For stage 5 differentiation, stage 4 differentiated cell aggregates werecontinued in either CMRL (Invitrogen/Gibco) or RMPI, Pen/Strep andGlutamax media, and 0.5×B27-supplement. In some experiments media wasalso supplemented with human serum (Valley Biomedical) or fetal bovineserum at concentrations ranging from 0.2-5% during stage 5.

Again, similar to the cell types from the stages 2-4, increasedexpression of genes associated with the specific cell type was observedas compared to the adherent plate controls. For example, increasedexpression levels of hormones insulin (INS), glucagon (GCG) andsomatostatin (SST) were observed. See FIG. 22, panel I (INS), panel J(GCG) and panel K (SST) at stage 5 (d15). Moreover, expression levels ofAFP and ZIC1, a gene associated with ectoderm, was significantly reducedin the hES-derived cell aggregates as compared to the adherent platecontrols. See FIG. 22, panel M (ZIC1) and panel N (AFP) at stage 5(d15). Thus, demonstrating that directed differentiation to producepancreatic endocrine cells by way of cell aggregates in suspensionculture removes ectoderm and visceral endoderm contaminants.

Production of hES-derived hormone expressing endocrine aggregate cellswas confirmed by flow cytometric analyses on Day 23 of the describedprotocol. Aggregates were initially formed at 140 rpm in 5 mL DMEM/F12,alternatively comprising knockout serum replacement (KSR;Gibco/Invitrogen compare 0063 for consistency) or xeno-free KSR(Invitrogen) and then differentiated at 100 rpm. Analysis of NKX6.1,Chromogranin A, insulin, glucagon and somatostatin protein expressionindicates that ES cell-derived aggregates are comprised of ˜20%NKX6.1+/Chromogranin A− pancreatic epithelium and ˜74% Chromogranin A+endocrine tissue. Moreover, 11% of the cells express insulin, 14%express glucagon and 11% express somatostatin. Of these, 68% of theinsulin+ cells are single positive, 70% of the glucagon+ cells aresingle positive and 52% of the somatostatin-positive cells are singlepositive. This degree of single hormone positivity exceeds the valuesdescribed for adherent cultures which were mostly polyhormonal cells.

To further evaluate the efficiency of aggregate differentiation tohormone expressing endocrine cells, cryosections of ES-derivedaggregates were examined for glucagon, insulin and somatostatinexpression using immunocytochemistry and confocal microscopy duringstage 5. Image analysis of cryosections at 20× indicates highlyefficient differentiation of aggregated cells to hormone positivity,with nearly all cells expressing glucagon, somatostatin or insulin.Also, in contrast to previous adherent culture experiments, a majorityof the cells in the aggregate appear to express a single hormone, asoccurs in vivo during development.

Example 19 Adherent Cultures from Various Stages can Form CellAggregates and Differentiate to Pancreatic Endoderm Type Cells

The following demonstrates that production of hES-derived cellaggregates can be initiated not just from pluripotent hESC but cellaggregates can be initiated directly into a differentiation media (day 0cell aggregates) as well as from differentiated or hES-derived cells,for example, cell aggregates can be produced from stages 1, 2, 4 and 5or hES-derived cells.

Day 0 Cell Aggregates

Cell aggregates produced on the first day (d0) of stage 1: Adherentpluripotent hESC were grown, manually or enzymatically passaged,disassociated, counted, pelleted and the pellet resuspended to a finalvolume of about 1×10⁶ cells/mL to 4×10⁶ cells/mL in differentiationmedia base containing RMPI, Pen/Strep and Glutamax media, and furthercontaining 100 ng/mL activin A, and 25 ng/mL-75 ng/mL Wnt3a, 0.2% of FBS(HyClone). This volume can be increased to >4×10⁶ cells/mL but mayrequire more frequent feeding. Sometimes DNase was included at aconcentration of 10-50 ng/mL. In some cases the ROCK inhibitor Y27632(Axxora) was added to the cell suspension to a final concentration of1-15 μM, typically 10 μM. Still in other cases about 1:2000 to 1:5000 ofITS (insulin/transferrin/selenium, Gibco) was added to the cultures.Both the Rho-kinase inhibitor and ITS were added to support cellsurvival. Resuspended cells were distributed equally into each well of alow binding 6-well dish substantially as described above, and placed onthe rotating platform at 100 rpm to 140 rpm overnight. During thisculturing period, cell aggregates of uniform size and shape were formed.Consequently, the higher density cultures effectively enriched orsubstantially enriched for PDX1-positive pancreatic endoderm orPDX-positive pancreatic progenitor type cells. Further details areprovided in Example 21.

Cell aggregates produced on d0 of stage 1, were then fed up to 1-2×daily with further differentiation media containing RMPI, Pen/Strep andGlutamax media, and further containing 100 ng/mL activin A and 0.2% ofFBS (HyClone) for the next 2-3 days. Subsequent steps (stages 2-5) ofthe protocol are substantially as described above for ES aggregates.

Stage 1—Day 2 to Day 3

Cell aggregates produced on d2-d3 of stage 1: Adherent hESC were grownand passaged substantially as described above and then differentiated tostage 1 substantially as described in D'Amour et al. 2006, supra.

Adherent cultures at the end of stage 1 (about d2 or d3 into thedifferentiation protocol; definitive endoderm type cells) were washed 1×with PBS−/− and disassociated to single cells with 2 mL of pre-warmedAccutase for about 2-5 minutes at 37° C. using a 1 mL or 5 mL pipet.Then 4 mL of 10% FBS in RMPI, Pen/Strep and Glutamax media was added andthe single cell suspension filtered through a 40 micron blue filter (BDBiosciences) into a 50 mL conical tube. The cells were counted andpelleted (centrifuged) substantially as described above.

The cell pellet was then resuspended in RMPI, Pen/Strep and Glutamaxmedia, further containing 2% FBS, plus DNase (50-100 μg/mL, RocheDiagnostics) and 100 ng/mL activin A. Alternatively the cell pellet wasresuspended in RMPI, Pen/Strep and Glutamax media, plus 2% FBS, andDNase (50-100 μg/mL), 25 ng-50 ng/mL KGF (R&D Systems). In someexperiments 5 μM SB431542 (Sigma Aldrich, Inc.) or 2.5 μM TGF-betaInhibitor IV (Calbiochem) was included with the KGF). In someexperiments Y27332 (10 μM) was included. Resuspended cells weredistributed equally into each well of a low binding 6-well dishsubstantially as described above, and placed on the rotating platform at100 rpm to 140 rpm overnight, during which time cell aggregates ofuniform size and shape were formed.

Cell aggregates produced at the end of stage 1 were then furtherdifferentiated. Subsequent steps (stages 2-5) of the protocol aresubstantially as described above for ES aggregates above in Examples 17and 18.

Stage 2—Day 5 to Day 6

Cell aggregates produced on d5-d6 at stage 2: Adherent hESC were grownand passaged substantially as described above and then differentiated tostage 2 substantially as described in D'Amour et al. 2006, supra. Forstage 2, adherent cells from stage 1 were briefly washed in PBS+/+ andthen further differentiated in RPMI supplemented with 2% FBS, Glutamax,penicillin/streptomycin, and 25 ng-50 ng/mL KGF (R&D Systems) for 3days. In some experiments 5 μM SB431542 (Sigma Aldrich, Inc.) or 2.5 μMTGF-beta Inhibitor IV (Calbiochem) was added during the first day ofstage 2.

Adherent cultures at the end of stage 2 (about d5 or d6 into thedifferentiation protocol; foregut type cells) were disassociated tosingle cells, counted and pelleted substantially as described above. Thecell pellet was then resuspended in differentiation media containingDMEM, Pen/Strep and Glutamax media, further containing 1×B27-supplementand DNase (50-100 μg/mL, Roche Diagnostics) and no FBS or 1-2% FBS or0.5%-10% human serum (hS) and either 1 μM to 2 μM retinoic acid (RA,Sigma) and 0.25 nM KAAD-cyclopamine (Toronto Research Chemicals); or 1μM to 2 μM retinoic acid, 0.25 nM KAAD-cyclopamine plus 50 ng/mL noggin(R&D systems); or 0.25 nM KAAD-cyclopamine plus 100 ng/mL noggin; or 100ng/mL noggin; or 0.2 μM to 0.5 μM RA and 0.25 nM KAAD-cyclopamine; or0.2 μM to 0.5 μM RA and 0.25 nM KAAD-cyclopamine plus 50 ng/mL noggin.In some experiments Y27332 (10 μM) was included.

Resuspended cells were distributed equally into each well, and placed onthe rotating platform at 100 rpm to 140 rpm overnight, during which timecell aggregates of uniform size and shape were formed.

The cell aggregates produced at the end of stage 2 were furtherdifferentiated on the rotating platform and fed 1-2× daily for 0-2additional days with DMEM, Pen/Strep and Glutamax media, furthercontaining 1×B27-supplement either 1 μM to 2 μM retinoic acid (RA,Sigma) and 0.25 nM KAAD-cyclopamine (Toronto Research Chemicals); or 1μM to 2 μM retinoic acid, 0.25 nM KAAD-cyclopamine plus 50 ng/mL noggin(R&D systems); or 0.25 nM KAAD-cyclopamine plus 100 ng/mL noggin; or 100ng/mL noggin; or 0.2 μM to 0.5 μM RA and 0.25 nM KAAD-cyclopamine; or0.2 μM to 0.5 μM RA and 0.25 nM KAAD-cyclopamine plus 50 ng/mL noggin.

Cell aggregates produced at the end of stage 2 were then furtherdifferentiated to stages 3, 4 and 5 substantially as described above.

Stages 4 and 5—Day 10 to Day 30

Cell aggregates produced on d10-d14 at stage 4: Again, adherent hESCwere grown and passaged substantially as described above and thendifferentiated to stage 2 substantially as described above and inD'Amour et al. 2006, supra.

For stage 3, adherent cells from stage 2 were further differentiated inDMEM, Pen/Strep and Glutamax media, further containing 1×B27-supplement,and either 1 μM to 2 μM RA and 0.25 nM KAAD-cyclopamine for 1 to 3 days.In other cases, 50 ng/mL noggin was added along with the RA andKAAD-cyclopamine. Alternatively, 0.2 μM to 0.5 μM of RA and 0.25 nM ofKAAD-cyclopamine was added to the media for just one day. Still, inother experiments no RA or KAAD-cyclopamine was added on any day. Atstage 4, cells were fed 1-2× daily with DMEM supplemented with Glutamax,penicillin/streptomycin, and 1×B27-supplement. Stage 4 cells can befurther differentiated to stage5 cells as already described in Examples17 and 18.

Adherent cultures at either stage 4 (about d10-d14 into thedifferentiation protocol; pancreatic epithelial and endocrine typecells) or stage 5 (about day 16 to day 30 into the differentiationprotocol; endocrine precursor and endocrine cells) were similarlydissociated into single cells, counted, and pelleted. The cell pelletwas then resuspended in DMEM CMRL supplemented Pen/Strep and Glutamax,and 1×B27-supplement and DNase (50-100 μg/mL, Roche Diagnostics) and0-2% FBS. In some experiments Y27332 (10 μM) was included whichsupported cell survival. Cells were equally distributed into 6-wellplates and placed on a rotating platform at 100 rpm to 140 rpm form 4hours to overnight substantially as described above.

Furthermore, the cell aggregates produced at stage 2 and at stage 5 asin Examples 17-19 were effectively enriched for pancreatic cell types ascompared with adherent plate cultures from which they were derived. Forexample, in one typical experiment cell aggregates produced at stage 2and analyzed by flow cytometry at stage 4 consisted of at least 98%pancreatic cell types (73% Chromogranin A positive endocrine cells and25% NR×6.1 positive pancreatic endoderm (PE)), and 2% non-pancreaticcell types; whereas the adherent plate cultures from which the cellaggregates were derived consisted of about 73% pancreatic cell types(33% Chromogranin A positive endocrine cells and 40% NR×6.1 positivePE), and 27% non-pancreatic cell types. Thus, aggregation at stage 2 caneffectively enrich for progenitors that give rise to pancreaticcell-types, and deplete for non-pancreatic cell types. Similarly, in atypical experiment, cell aggregates produced at stage 5 and analyzed byflow cytometry consisted of at least 75% Chromogranin A positiveendocrine cell types, whereas the adherent plate culture from which thecell aggregates were derived consisted of about 25% Chromogranin Apositive endocrine cell types. Hence, aggregation at stage 5 caneffectively enrich pancreatic endocrine cells.

The methods described herein, therefore, provide methods for improvingnot only efficiency of directed-differentiation of hESC in cellaggregate suspensions, but also provides methods for reducinghES-derived pancreatic cell types (or aggregates) having contaminantpopulations (e.g. ectoderm, trophectoderm, visceral endoderm, andextra-embryonic endoderm) and at the same time enrichment of pancreaticcell types (e.g. pancreatic endoderm and endocrine cells).

Example 20 Cell Density Effects hES Cell Differentiation Outcome

The following demonstrates that variations in cell densities effectdifferentiation outcomes within a given media and growth factorcondition. The differentiation efficiency outcomes which result fromadjustments in cell density reflects varying concentrations ofendogenously produced signaling molecules and the concentrationdependent affect of these molecules in influencing cellulardifferentiation.

Human ES cell aggregates and hES-derived cell aggregates, including d0cell aggregates produced directly in differentiation media, weregenerated substantially as described above. After about five (5) days ofdifferentiation through stages 1 and 2, the differentiating cellaggregates were pooled and re-aliquoted into individual wells atdifferent seeding densities, e.g., a 28 mL suspension of foregutendoderm stage cell aggregate suspension was seeded or re-aliquoted at4, 6, 8 or 10 mL per well (a 2.5-fold range of cell densities). Thiscell distribution was carried out in duplicate and one set of wells wasfed with a stage 3 media (DMEM/PenStrep/Glutamax+1% B27 supplement(vol/vol)+0.25 uM KAAD-cyclopamine+3 nM TTNPB) containing noggin at 50ng/mL and the other set of wells contained noggin at 25 ng/mL. Stage 3proceeded for 3 days with daily media exchange. Cell samples were takenin duplicate for real-time QPCR analysis at the end of the three days ofstage 3 (or about day 8) and again at after stage 4 (or about day 14).

The cell density and noggin concentration used during stage 3 haddifferent effects on the expression of those genes which are indicativeof pancreatic endoderm progenitors and/or endocrine progenitors orprecursors. Briefly, there is a linear relationship between increase incell density and a corresponding increase in pancreatic progenitor celltypes (e.g., pancreatic endoderm, pancreatic epithelium, PDX1-positivepancreatic endoderm). For example, after stage 3 (or day 8), an increasein cell density had a corresponding increase in the cell numbers ofpancreatic progenitors as indicated by enhanced gene expression of PDX1and NKX6-1. See FIGS. 24A & 24B. In contrast, there was an inverserelationship between increase in cell density and a correspondingreduction in endocrine progenitor cell types after stage 4 (or day 14).For example, as the cell density decreased there was reduced expressionof at least NGN3 and NKX2-2 after stage 3 (or day 8). See FIGS. 24C &24D.

Yet, lower concentrations of noggin (e.g., 25 ng/mL) at any given celldensity resulted in reduced endocrine progenitor cell types as indicatedby reduced expression of NGN3 and NKX2-2. See FIGS. 24C & 24D. This celldensity independent effect of noggin in the cell cultures suggests thatendogenously produced BMP signals from the cells are antagonized by theexogenously added noggin. The impact of endogenously produced signals ondifferentiation outcome is likely not limited to just BMP, but othergrowth factors and/or agents secreted by the cells into the medium canhave similar or contrasting effects, alone or in combination withexogenous growth factors and/or agents.

Example 21 Optimization of Cell Aggregate Suspension Cultures toGenerate Enriched Pancreatic Endoderm or Endocrine Cell Types

The cell composition of hES-derived cell aggregate populations isoptimized for certain cell types by controlling the concentration ofvarious growth factors and/or agents. The pancreatic cell compositionsdescribed herein were hES-derived cell aggregate suspensions which weremade from single cell suspension cultures, which were derived from hEScell adherent cultures, d0 cell aggregates (cell aggregates initiatedfrom hES adherent cultures but directly into a differentiation media andnot a pluripotent stem cell media), or from hES-derived cell adherentcultures at various stages of differentiation substantially as describedin the previous examples. During stage 4, cell aggregates were exposedto different concentrations of the factors: NOGGIN (N), KGF (K), FGF10(F), and EGF (E). The cell composition of the differentiated hES cellaggregates was assessed by flow cytometry analysis using a panel ofmarkers including CHGA, NKX6.1, and PDX1. The total percentage ofendocrine cells, pancreatic endoderm cells, PDX1+ endoderm cells, andnon-pancreatic cells in any cell population is shown in Table 6.

The data in Table 6 demonstrates that by controlling the concentrationand ratios of certain growth factors, the resulting composition can beoptimized for certain cell types. For example, the percentage ofpancreatic endoderm type cells was increased as compared to endocrinetype cells by lowering the concentration of KGF and EGF (e.g.,K(25)E(10) and 71% vs. 22.1%). In contrast, high concentrations of KGFand EGF and inclusion of Noggin and FGF10 (e.g., N(50)F(50)K(50)E(50))decreased the number of pancreatic endoderm type cells, the total numberbeing comparable to that of endocrine type cells (e.g., 39.6% vs.40.1%). Noggin and KGF in higher concentrations (e.g., N(50)K(50)) ornot adding growth factor increased the population of endocrine typecells in the resulting population as compared to pancreatic endodermcell types. Also, the percentage of non-pancreatic cell types (i.e. nonPDX1-positive type cells) can be significantly reduced by reducing thelevels of KGF and EGF (e.g., K(25)E(10); 1.51%) or not adding any growthfactor (1.53%).

Thus, Table 7 clearly demonstrates that at least varying theconcentrations of different growth factors in the culture medium atcertain stages of differentiation (e.g., stage 4) significantlyincreases and/or decreases certain populations of pancreatic endoderm,endocrine, PDX1-positive endoderm or non-pancreatic cell types.

TABLE 7 The effects of growth factors on cell composition PancreaticPDX1+ Non- Endoderm Endoderm Pancreatic CHGA- CHGA− CHGA− AggregationFactors in Stage 4 Endocrine NKX6.1+ NKX6.1− NKX6.1+/− Stage Media(ng/mL) CHGA+ PDX1+ PDX1+ PDX1− Total ESC K(25)E(10) 22.1 71.0 3.0 4.0100.1 ESC K(25)E(10) 29.0 67.1 2.37 1.61 100.0 Stage 1 Day 0 K(25)E(10)25.4 68.9 2.87 2.01 99.2 Stage 1 Day 0 N(50)K(50)F(50)E(50) 40.1 39.613.30 6.85 99.9 Stage 1 Day 0 None added 69.4 27.4 1.46 1.53 99.8 Stage1 Day 0 N(50)K(50) 52.2 30.4 13.9 3.48 99.9 Stage 1 Day 0 K(25)E(10)38.8 50.8 2.17 8.22 100.0 Stage 2 Day 5 N(50)K(50) 42.3 42.3 12.2 3.2099.9 Stage 2 Day 5 K(25)E(10) 28.3 59.4 7.36 4.97 100.0

Still other methods exist for enriching or purifying for particularhES-derived cells types as described in U.S. patent application Ser. No.12/107,020, entitled METHODS FOR PURIFYING ENDODERM AND PANCREATICENDODERM CELLS DERIVED FROM HESC, filed Apr. 8, 2008, which is hereinincorporated in its entirety by reference. This application describesmethods for enriching various hES-cell types including all the celltypes resulting in each of stages 1, 2, 3, 4 and 5 as described inD'Amour et al. 2005, supra and 2006, supra. The application uses variousantibodies including but not limited to CD30, CD49a, CD49e, CD55, CD98,CD99, CD142, CD165, CD200, CD318, CD334 and CD340.

Methods for enriching the hES-derived cells or cell aggregates are notlimited to methods employing antibody affinity means, but can includeany method which is available to or will be well known to one ofordinary skill in the art that allows for enrichment of a certain celltype. Enrichment can be achieved by depleting or separating one celltype from the another cell type or culture.

Example 22 Cell Aggregate Suspensions of Pancreatic Endoderm Mature InVivo and are Responsive to Insulin

To demonstrate that the methods for making and manufacturing cellaggregate suspensions as described herein provides pancreatic progenitorcells which function in vivo, the above hES-derived cell aggregates inExamples 17-21 (e.g., PDX1-positive endoderm, pancreatic endoderm,pancreatic epithelium, endocrine precursors, endocrine cells, and thelike) have been transplanted into animals. Methods of transplantationinto normal and diabetic-induced animals, determination of in vivoglucose responsiveness of the animals and therefore insulin productionof the mature transplanted cells in vivo, were performed substantiallyas described in Kroon et al. 2008, supra and U.S. patent applicationSer. No. 11/773,944, entitled METHODS OF PRODUCING PANCREATIC HORMONES,filed Jul. 5, 2007, which are incorporated herein in their entireties.Substantially similar levels of human C-peptide were observed in thesera of these animals at similar time periods as indicated in Kroon etal. 2008, supra and U.S. patent application Ser. No. 11/773,944, supra.

The methods, compositions, and devices described herein are presentlyrepresentative of preferred embodiments and are exemplary and are notintended as limitations on the scope of the invention. Changes,alternatives, modifications and variations therein and other uses willoccur to those skilled in the art which are encompassed within thespirit of the invention and are defined by the scope of the disclosure.Accordingly, it will be apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

It is appreciated that certain features of the invention, which are, forclarity described in the context of the separate embodiments, may alsobe provided in combination in a single embodiment. For example, methodsfor making hES-derived cell aggregates in suspension can be generatedand optimized to produce any endoderm lineage cell type, e.g., apancreatic lineage type cell, a liver lineage type cell, an epitheliallineage type cell, a thyroid lineage cell and a thymus lineage cell, andtherefore is not limited to the hES-derived cell types specificallydescribed therein. Conversely, various features of the invention, whichare, for brevity, described in the context of a single embodiment, mayalso be provided separately or in any suitable subcombination. Forexample, it is apparent to one skilled in the art that the describedmethods for generating hES and hES-derived cell aggregates from adherentplate cultures or from suspension, from undifferentiated adherent platecultures or from suspension, and from differentiated adherent platecultures or from cell aggregates in suspension are just exemplary butthat a combination of the methods may also be employed.

Example 23 Cryopreservation and Banking of Human Pluripotent Stem Cellsand Pancreatic Progenitor Cells

Adherent hES cell cultures were harvested according to the describedpassaging protocol described in Example 24, pooled and counted. Cellpellets were re-suspended in pre-warmed about 50% hESC culture medium(without growth factors)/50% human serum. An equal volume of about 80%hESC culture medium (without growth factors)/20% DMSO was addeddrop-wise, with swirling. 1 mL of cells was distributed to 1.8 mLcryovials for freezing at −80° C. in Nalgene Mr. Frosty containers forabout 24 hours, before transferring to liquid N₂. Substantially similarmethods were performed under cGMP.

The above methods describe cryopreservation of pluripotent ordifferentiable stem cells. Cryopreservation of cells differentiated frompluripotent stem cells, for example, pancreatic progenitor cells werepreviously described in detail in U.S. patent application Ser. No.12/618,659, entitled ENCAPSULATION OF PANCREATIC LINEAGE CELLS DERIVEDFROM HUMAN PLURIPOTENT STEM CELLS, filed Nov. 13, 2009, whichapplication and related applications are incorporated herein byreference in their entireties.

Example 24 Adherent Culture, Passaging & Expansion of UndifferentiatedHuman Pluripotent stem cells in various culture vessels including rollerbottles

A major bottleneck for manufacture of an cell aggregate-based celltherapy product, in particular one derived from human pluripotent stemcells, is the formation of the 3-dimensional cellular aggregate insuspension. Specifically, the bottleneck is taking monolayer adherentpluripotent stem cells and converting them into pluripotent stem cellaggregates, i.e. suspension aggregate cultures. As described above, hEScell aggregates in 6-well trays can be formed, for example, andsubsequently differentiated in either 6-well trays or other culturevessel formats, e.g. bioreactors, bottles and the like. The embodimentsof the invention described in the following Examples, provides methodsfor growth, passage and expansion of pluripotent stem cells as cellaggregates in suspension as well as for differentiating the cellaggregates in roller bottles. See Example 25.

Culture and Expansion of Human Pluripotent Stem Cells.

Upon thaw, or at regular passaging, dissociated hESC were plated at50,000 or 33,000 cells/cm² for three and four day growth cycles,respectively, in different cell culture vessels. hESC growth media (XFHA) consisted of DMEM/F12 containing G1utaMAX, supplemented with 10% v/vof Xeno-free KnockOut Serum Replacement, 1% v/v non-essential aminoacids, 0.1 mM 2-mercaptoethanol, 1% v/v penicillin/streptomycin, 10ng/mL heregulin-1β and 10 ng/mL activin A. On the day of plating only(one day treatment), cell attachment was facilitated by including about10% (vol/vol) of non-heat inactivated human AB serum (Valley Biomedical)simultaneously with the addition of xeno-free culture medium asdescribed previously. A standardized plating volume of 0.2 mL/cm² wasused for different tissue culture plates, T-flasks and cell factories asdescribed at least in Table 8 below. The volume of growth media used wasincreased for each additional day of feeding and is also indicated inTable 8.

TABLE 8 Culture & Expansion of adherent hES cell Cultures Vessel Triple2-stack Type 60 mm T75 T175 T175 1272 5-stack S.A. 19.6 cm² 80 cm² 175cm² 525 cm² cm² 3180 cm² Plating 4 16 35 105 260 650 dl 5.5 22 50 150350 875 d2 7 28 60 180 450 1100 d3 8.5 35 80 240 550 1350 Volumes in mL;SA: surface area.

Passaging Human Pluripotent Stem Cells.

On the day of passaging, cultures were fed with fresh growth medium andcultured for 4-8 hours before dissociation. Cultures were washed withPBS (Life Technologies) and dissociated for 6 minutes at about 37° C.using pre-warmed ACCUTASE (Innovative Cell Technologies). In someexperiments the ACCUTASE was added, and then immediately aspirated (i.e.less than 4-6 minutes), such that cell dissociation was achieved in theresidual reagent, at a minimal working volume, which is preferred whenworking with certain culture vessels, including cell factories, tominimize the number of media exchange steps. After exposure to ACCUTASE,3× volume of cold hESC media (without heregulin or activin) was addedand the cells were dissociated and collected. Dissociated cells weregently collected using 3× volume of cold hESC media (without heregulinor activin), counted using a ViCell automated cell counter (BDBiosciences), or a hemocytometer, centrifuged for 5 minutes at 200×g andthe cell pellet re-suspended in fresh growth medium at 1−10×10⁶ cells/mLfor subsequent plating under the same culturing conditions.

Table 8 describes a variety of culture vessels and media volumes thathave been used, however, the skilled artisan will appreciate that otherculture vessels not specifically described can be used for growth,passage, and expansion of human pluripotent stem cells based on thedetailed descriptions described herein. For example, see Example 25 formethods of suspension differentiation in a roller bottle.

In some studies, the passaged hESCs were added to new, uncoated 6-welltray culture vessels, without cell attachment, and rotated to formaggregates, as described above. Typically, StemPro® hESC SFM medium(Life Technologies) supplemented with 10 ng/mL heregulin-1β and 10 ng/mLactivin A, or XF-HA medium, was used for suspension culture of hESC.Cell culture was performed in humidified incubators at 37° C. and 8%CO₂.

In order to test the aggregation of pluripotent stem cells in a rollingbottle format, single cell suspensions of 10⁶ hESC/mL preparedsubstantially as described above for hESC aggregation from adherent hESCcultures or directly from vials of frozen cells that were thawed, washedand suspended in culture medium, were placed in different vessels, eachwith a tubular shape which could be rotated while placed on its side. 50mL tubes containing 10 mL cell suspension and 150 mL bottles containingeither 30 mL or 120 mL of cell suspension were placed in a hybridizationoven at about 37° C. and rotated at about 5 rpm. Rotation was achievedusing a built-in, variable speed, mechanical bottle rotator. In theinitial studies, the ovens were not gassed with CO₂. For purposes ofcell aggregation control, simultaneous studies were performed using6-well trays. Based on aggregate diameter and morphology, pluripotentstem cell aggregates were formed successfully in the rolled bottleformat and the control 6-well tray format. Pluripotent stem cellaggregates were also formed in previous experiments using plastic jars.These studies demonstrated that aggregation could proceed effectively ina vessel format that was completely different from rotational culture in6-well trays, with respect to speed, vessel shape and fluid dynamics.Furthermore, this rolling bottle format would likely be scalable withoutsubstantial optimization of the methods described herein, to bypass acritical bottleneck area.

Example 25 Pluripotent Stem Cell Aggregation and Differentiation inRoller Bottles

Human ES cells (Example 24) were aggregated in 150 mL bottles, anddifferentiated to pancreatic progenitors (or PEC) using Applicant'sstages 1-4 differentiation protocol, as described above. 150 mL rollerbottles were seeded with 120 mL cell suspension of either 1×10⁶cells/mL, or 2×10⁶ cells/mL cell densities in StemPro® hESC SFM media orXF HA media; see Table 9. Stages 1-4 media conditions were substantiallyas that previously described (see Schulz et al. (2012) supra), which aresummarized in Table 9. Rotation speeds of about 5 rpm, 8 rpm, 10 rpm or12 rpm were tested throughout the hESC aggregation and Stages 1-4differentiation and gassing with CO₂ was not incorporated into theincubator. Gassing with CO₂ may depend on what caps are used with theroller bottles, e.g., plug caps, vented or un-vented caps. FIG. 25 showsthe average diameter size of the cell aggregates formed during rollerbottle aggregation and differentiation. Each box plot shows the minimum,maximum, 2nd and 3rd quartile, and median of the initialundifferentiated (d0) and differentiating cell aggregates (d2, d5, d8and d12). Cell aggregate diameters were measured for both conditions.The average diameter of the cell aggregates initially formed was largerwhen the cultures were seeded at about 1×10⁶ cell/mL as compared 2×10⁶cell/mL. However, at later stages of differentiation (e.g. Stages 3-4)the diameter sizes were comparable and indistinguishable. FIG. 25 alsoshows that there were no substantial differences between thedifferentiated cell aggregates that formed in the roller bottles andthose that formed during previous suspension differentiation experimentsperformed in 6-well trays. Differentiation in the roller bottles alsoshowed the typical expansion and contraction of aggregate diameter aspreviously observed in cultures performed in 6-well trays, bioreactorsand the like (FIG. 25). The aggregate diameter was independent of theinitial cell density and independent of the initial hESC orundifferentiated cell growth media composition. Briefly, the cellaggregates expanded during stages 1 and 2 (FIG. 25, d0 and d2),contracted during stage 3 (FIG. 25, d5), and expanded again during stage4 (FIG. 25, d8 and d15). Throughout the differentiation, the cellaggregates did not show overt agglomeration (e.g., large aggregates of300 microns or more) or shear-destruction.

TABLE 9 Media Conditions for Stages 1-4 Differentiation in 6-Well Traysand Roller Bottle Time point Stage Roller Bottle 6-well tray Speed (day)(1-4) Media Condition Speed (rpm) (rpm) d(−1) hESC XF HA, SP 5, 8, 10 or12 95 Aggregation d0 1 r0.2FBS-ITS1:5000 A100 5, 8, 10 or 12 95 d1r0.2FBS-ITS1:5000 A100 5, 8, 10 or 12 95 d2 2 r0.2FBS-ITS1:1000 K25 5,8, 10 or 12 95 d3 r0.2FBS-ITS1:1000 K25 5, 8, 10 or 12 95 d4r0.2FBS-ITS1:1000 K25 5, 8, 10 or 12 105 d5 3 db-CTT3 N50 5, 8, 10 or 12105 d6 db-CTT3 N50 5, 8, 10 or 12 105 d7 db-CTT3 N50 5, 8, 10 or 12 105d8 4 db-N50 K50 E50 5, 8, 10 or 12 105 d9 db-N50 K50 E50 5, 8, 10 or 1295 d10 db-N50 K50 E50 5, 8, 10 or 12 95 d11 db-N50 K50 E50 5, 8, 10 or12 95 d12 db-N50 K50 E50 5, 8, 10 or 12 95 XF HA, DMEM/F12 containingGlutaMAX, supplemented with 10% v/v of Xeno-free KnockOut SerumReplacement, 1% v/v non-essential amino acids, 0.1 mM 2-mercaptoethanol,1% v/v penicillin/streptomycin (all from Life Technologies), 10 ng/mLheregulin-1β (Peprotech) and 10 ng/mL activin A (R&D Systems); SP,StemPro ® hESC SFM (Life Technologies); r0.2FBS: RPMI 1640 (Mediatech);0.2% FBS (HyClone), 1x GlutaMAX-1 (Life Technologies), 1% v/vpenicillin/streptomycin; ITS: Insulin-Transferrin-Selenium (LifeTechnologies) diluted 1:5000 or 1:1000; A100: 100 ng/mL recombinanthuman Activin A (R&D Systems); W50: 50 ng/mL recombinant mouse Wnt3A(R&D Systems); K25: 25 ng/mL recombinant human KGF (R&D Systems); IV:2.5 μM TGF-β RI Kinase inhibitor IV (EMD Bioscience); db: DMEM HIGlucose (HyClone) supplemented with 0.5x B-27 Supplement (LifeTechnologies), 1x GlutaMAX-1 and 1% v/v penicillin/streptomycin; CTT3:0.25 μM KAAD-Cyclopamine (Toronto Research Chemicals) and 3 nM TTNPB(Sigma-Aldrich); N50: 50 ng/mL recombinant human Noggin (R&D Systems);K50: 50 ng/mL recombinant human KGF (R&D Systems); E50: 50 ng/mLrecombinant human EGF (R&D Systems); 5, 8, 10, 12 rpm rotation speedwere performed at either the hESC aggregation, at stages 1-4differentiation, or both.

To examine gene expression throughout stages 1-4, Q-PCR was used toanalyze the differentiations performed with 1×10⁶ cell/mL vs. 2×10⁶cell/mL starting cell densities (FIG. 26). Although only certain genesare shown in FIG. 26, Applicant has previously described expression andnon-expression of many genes in each of stages 1-4 in extensive detail.See e.g., U.S. Pat. Nos. 8,211,699, METHODS FOR CULTURING PLURIPOTENTSTEM CELLS IN SUSPENSION USING ERBB3 LIGANDS, issued Jul. 3, 2012;7,958,585, PREPRIMITIVE STREAK AND MESENDODERM CELLS, issued Jul. 26,2011; 7,510,876, DEFINITIVE ENDODERM (CYTHERA.045A), issued on Mar. 31,2009; 7,541,185, METHODS FOR IDENTIFYING FACTORS FOR DIFFERENTIATINGDEFINITIVE ENDODERM, issued Jun. 2, 2009; 7,625,753, EXPANSION OFDEFINITIVE ENDODERM, issued Dec. 1, 2009; 7,695,963, METHODS FORINCREASING DEFINITIVE ENDODERM PRODUCTION, issued Apr. 13, 2010;7,704,738, DEFINITIVE ENDODERM, issued Apr. 27, 2010; 7,993,916, METHODSFOR INCREASING DEFINITIVE ENDODERM PRODUCTION, issued Aug. 9, 2011;8,008,075, STEM CELL AGGREGATE SUSPENSION COMPOSITIONS AND METHODS OFDIFFERENTIATION THEREOF, issued Aug. 30, 2011; 8,178,878, COMPOSITIONSAND METHODS FOR SELF-RENEWAL AND DIFFERENTIATION 1N HUMAN EMBRYONIC STEMCELLS, issued May 29, 2012; 8,216,836, METHODS FOR IDENTIFYING FACTORSFOR DIFFERENTIATING DEFINITIVE ENDODERM, issued Jul. 10, 2012;7,534,608, METHODS OF PRODUCING PANCREATIC HORMONES, issued May 19,2009; 7,695,965, METHODS OF PRODUCING PANCREATIC HORMONES, issued Apr.13, 2010; 7,993,920 METHODS OF PRODUCING PANCREATIC HORMONES, issuedAug. 9, 2011; 8,129,182, ENDOCRINE PRECURSOR CELLS, PANCREATIC HORMONEEXPRESSING CELLS AND METHODS OF PRODUCTION, issued Mar. 6, 2012; U.S.patent application Ser. Nos. 11/875,057, METHODS AND COMPOSITIONS FORFEEDER-FREE PLURIPOTENT STEM CELL MEDIA CONTAINING HUMAN SERUM, filedOct. 19, 2007; 12/618,659, ENCAPSULATION OF PANCREATIC LINEAGE CELLSDERIVED FROM HUMAN PLURIPOTENT STEM CELLS, filed Nov. 13, 2009; whichare all incorporated herein by reference in their entireties. Only afterobtaining a high degree of confidence in the differentiation methods didApplicant select a smaller set of markers as the signature markers toindicate and identify the various stages 1-4 cell populations as shownin FIG. 26.

FIG. 26 shows that cell aggregates differentiated in rolling bottleformats expressed the typical signature markers expected at each stageof differentiation, in agreement with differentiation studies performedsimultaneously in 6-well trays (control, FIG. 26). Similarly, absence ofexpression of signature markers was observed where expected. Forexample, pluripotent stem cell markers such as OCT4 and Nanog werepresent at d0 for all culture conditions (FIG. 26A). Similarly, therewas transient up-regulation of the mesendodermal markers MIXL1 and Eomesin both the roller bottle differentiations and that the 6-well traydifferentiationss (FIG. 26, compare the black bar, blacked hatched bar,grey bars, respectively). Stage 2 cells produced definitive endoderm,which expresses SOX17 and HNF3β [FOXA2], similarly to that observed forstage 2 type cells differentiated in 6-well trays. Stage 4 type cellsexpressed PDX1 and NKX6.1 similarly to that observed for culturesdifferentiated in 6-well trays (FIG. 26, compare black bar, blackedhatched bar, grey bars, respectively). Lastly, expression of markers foroff-target lineages was not substantially different in culturesdifferentiated in 6-well trays and 150 mL rolling bottles, indicatingthat control of differentiation remained tight in the rolling format.These markers included ZIC1 (ectoderm lineage), early expression PAX6(neuronal lineage), SOX7 (extraembryonic endoderm), CDX2(trophectoderm), and early expression of AFP (yolk sack).

TABLE 10a pPSC Differentiation in Large Roller Bottles Caps, VentedAggre- (V); % gate Bottle Not- pellet Incorpo- dia- Bottle SurfaceAggregation Vented volume ration meter Volume area Volume (NV) d0 d0(μm) 1200 mL 490 cm² 275 mL V 1050 μL 79.6 177 ± 23 1200 mL 490 cm² 275mL NV 1090 μL 78 167 ± 21 2275 mL 850 cm² 580 mL V 2300 μL 76 171 ± 266-well tray —  33 mL/tray — ~105 ~75 136 ± 15 μL/tray

Once it was demonstrated that pPSCs could be aggregated in a rollerbottle format on a small scale (Example 25), larger cultures wereprepared in order to demonstrate the practical scalability ofaggregating and differentiating hESC to PEC in roller bottles.Experiments using CyT49 hESC were perforemd as indicated in Table 10.Human ESCs were aggregated in StemPro® hESC SFM medium, Otherpluripotent stem cell media, for example XF HA media with and withouthuman serum albumin (HAS), was used in other experiments (data notshown). Day 0 (d0) hESC aggregates formed effectively in each condition.The experimental cultures summarized in Table 10 were rotated at 8 rpm.In other experiments, aggregation at 5, 10 and 12 rpm was utilized andhESC aggregates formed with similar morphology and diameters as thoseobserved in 6-well trays and roller bottles at 8 rpm (data not shown).In some instances where hESC aggregation was performed at the lowerrange of rotation speed, e.g. 5, 6, and 7 rpm or with StemPro® hESC SFM(Life Technologies) media, an increase in agglomeration of the cellaggregates was observed (i.e., structures greater than 300 μm; data notshown). Some experiments were performed using vented bottle caps (V)while others did not have vented bottle caps (NV). Vented did not make asubstantial difference on the differentiation process, nor did it appearto affect the proper specification of hESCs as determined by qPCR geneexpression analysis (FIG. 26). In summary, hESC aggregation in rollerbottles can be accomplished over a range of rotation speeds, (e.g.,between about 5 to 12 rpms), with various pluripotent stem cell culturemedia, and in vented or not vented bottle caps, and in CO₂ gassed orun-gassed incubators. These different factors do not appear tosubstantially change the morphology, shape and average diameter size ofthe aggregates.

Example 27 Scaled Differentiation of Stem Cell Aggregates in RollerBottles

Human ESC aggregation was again performed using 1200 mL or 490 cm²roller bottles substantially as described above in Examples 25 and 26.Because the hESC aggregates in each roller bottle aggregation lookedconsistent and similar (e.g. morphology and diameter size), theaggregates were pooled, pelleted, and the pellet of aggregatesdistributed between 1200 mL or 490 cm² roller bottles fordifferentiation according to Table 11. The total volume of the hESCaggregate pellet was approximately 4400 μL and was redistributed to4×1200 mL bottles (490 cm²) for differentiation according to Table 11.Differentiation was performed as described above in Table 9. Thedifferentiating cultures exhibited similar morphologies, except minoramounts of agglomeration was observed in bottles, which were rotated atthe lower rotation speeds (data not shown). However, aggregate pelletvolumes by day 12 (Stage 4) were similar for all conditions tested, andall cell pellets recovered were about a 1:1 yield as compared to thestarting pellet volume in each bottle (˜1100 μL). See Table 11.

Aggregation of pPSC is not limited to hESCs. Human iPSC were tested in asubstantially similar manner as that described above for hESCs, underconditions listed in Table 11. Human iPSC-482c7 (Cellular DynamicsInternational Inc. Madison, Wis., USA) were aggregated in a 490 cm²roller bottle. The total starting volume of the hiPSCs was only about 25mL at 1×10⁶ cells/mL. The aggregation platform is sufficiently robust toperform well over a range of cell volumes and even when there is agreater disparity between the starting volume and the larger 490 cm²roller bottle. The human iPSC cell aggregates appeared morphologicallysimilar to hESCs aggregated in 6-well trays or roller bottles asdescribed above, i.e., iPSC aggregate sizes ranges from about 100 toabout 300 microns with no apparent agglomeration. Also, as describedpreviously in U.S. patent application Ser. No. 12/765,714, entitled CELLCOMPOSITIONS DERIVED FROM DEDIFFERENTIATED REPROGRAMMED CELLS, filedApr. 22, 2010, which is incorporated herein in its entirety, the use ofRho kinase inhibitors improved pPSC aggregate differentiation; however,since the '714 application did not show hESC aggregation (onlydifferentiation), that application did not show that use of for example10 μM Y-27632 in the starting pPSC culture would also improve pPSCaggregation. Because hESC aggregation can be performed in a rollerbottle, cell aggregate differentiation in roller bottles can beperformed as well and substantially as described in detail in the '714application or related application, or substantially as described hereinand according to Table 9.

Additionally, to demonstrate the integrity of hESC aggregates, hESCaggregates were first formed in roller bottles and then subsequentlydifferentiated in 6-well trays. This was performed using hESC aggregatesfirst formed in roller bottles under a variety of conditions, e.g. atdifferent rotation speeds and with different pluripotent stem cellmedia. Differentiation of roller bottle hESC aggregates in 6-well trayswas comparable in aggregate shape and diameter, and cell morphology tothat observed when hESC aggregates were first formed in 6-well trays(control; data not shown). Hence, the integrity of the hESC aggregatesin roller bottles is substantially unchanged due to the format change.

To demonstrate that scaling using roller bottles does not compromiseincorporation of cells into aggregates, cell counting of the live,unincorporated cells following aggregate formation was performed at d0(i.e. 18-24 hours after the initiation of aggregations), confirming thatabout 75-80% of the input cells were incorporated into theundifferentiated hESC aggregates, which is comparable or better thanthat observed in 6-well trays (about 75%). See Schulz et al. 2012,supra, FIG. 54, S6. The percentage of incorporation may vary dependingon the pluripotent stem culture media used since studies using XF HAmedia provided a range of incorporation from about 50% to about 77%(data not shown).

Q-PCR analysis of the differentiation process according to Tables 11 and9, demonstrated that appropriate lineage specification occurred at eachstage up to formation of PEC. See FIG. 27A-D. The four cultures aslisted in Table 11 each exhibited down-regulation of markers ofpluripotency (e.g., OCT4, Nanog) and transient up-regulation of markersof mesendoderm (e.g. MixL1, Eomes). See FIG. 27A. Markers of definitiveendoderm (e.g., SOX17 and HNF313) were expressed from day 2 as expected,followed by other endodermal and pancreatic markers (e.g., HNF13, PDX1,NKX6-1, PTF1A, NGN3, and NKX2-2). See FIG. 27B. Importantly, markersindicative of off-target lineages (non-endodermal or non-pancreaticlineages) were not elevated as compared to the 6-well tray controldifferentiation (e.g., PAX6, SOX7, CDX2, AFP and ZIC1), hence tightcontrol of pancreatic specification was maintained in these scaleddifferentiation experiments. See FIG. 27C. For example, AFP expression,which typically indicates the presence of a minor off-target populationthat occurs sporadically in some differentiations, was low in the E-welltray control. See FIG. 27D and Schulz et al. 2012, supra. AFP expressionwas even lower in the roller bottle cultures, potentially indicating areduction in this minor population in the d12 aggregates. See FIG. 27D.

To assess the cellular compositions of pancreatic cell culturesdifferentiated from hESC with the multistep (Stages 1-4) protocol, flowcytometry analysis based on a combination of co-staining was usedsubstantially as that previously described in Kelly et al. 2011, NatureBiotech 29:750-56; D'Amour et al. 2005, supra; Kroon et al. 2008 supra,Schulz et al. 2012, supra, which are herein incorporated by reference intheir entireties. Schulz et al. 2012, supra for example showed extensiveflow cytometry analysis and PEC cellular composition for at least 37differentiation runs (FIG. 2). Schulz et al. described the cellularcomposition of PEC as consisting of: About 26-36%CHGA⁻/NKX6-1⁺/PDX1^(+/−), about 46-56% CHGA⁺/NKX6-1^(+/−)/PDX1^(+/−)(poly-hormonal endocrine cells), about 10-15% CHGA⁻/NKX6-1⁻/PDX1⁺(PDX1-only endoderm cells) and less than 3% CHGA⁻/NKX6-1⁻/PDX1⁻(residual or triple negative cells). See Schulz et al. 2012, supra FIG.2C.

Similarly, flow cytometry analysis was performed on stage 4 (day 12) PECcultures differentiated in a roller bottles as indicated in Table 11.The PEC composition was consistent with that previously described inSchulz et al. above and the exact percentages as well as the averagefrom the four roller bottle conditions is shown in Table 12. The PECfrom these scaled roller bottle differentiations showed about 40%CHGA⁻/NKX6-1⁺/PDX1^(+/−), about 43% CHGA⁺/NKX6-1^(+/−)/PDX1^(+/−) (orpolyhormonal endocrine cells), about 10% CHGA⁻/NKX6-1⁻/PDX1⁺ (orPDX1-only endoderm cells), and about 2% CHGA⁻/NKX6-1⁻/PDX1⁻ (or residualcells; or triple negative cells). Flow cytometry analysis of the PECcomposition also indicates that neither the different rotation speeds of5 and 8 rpm nor the type of vented or not-vented bottle caps had anapparent effect on PEC cell composition. As described above and based onQ-PCE analysis, these conditions did not appear to effect the lineagespecification of Stages 1-4 cells en route to PEC either (FIG. 26).Therefore, the above studies and analyses confirmed the effectiveness ofhESC aggregation and differentiation to PEC in scalable rolling bottleformat.

TABLE 11 pPSC Aggregation in Large Roller Bottles Bottle Stage 4,Surface area Aggregation Caps, Vented (V); Day 12 (1200 mL) VolumeNot-Vented (NV) Speed pellet volume 490 cm² 275 mL V 5 rpm 1000 μL 490cm² 275 mL NV 5 rpm 1100 μL 490 cm² 275 mL V 8 rpm  800 μL 490 cm² 275mL NV 8 rpm 1200 μL

TABLE 12 PEC Cell Composition from Roller Bottle after Stages 1-4Differentiation CHGA− CHGA− NKX6.1− CHGA+ CHGA− NKX6.1− PDX1− (Poly-NKX6.1+ PDX1+ (Residual- hormonal PDX1+ PDX Triple Endocrine) or − only)Negative) RB- A 490 cm² V 8 rpm 40.2 41.0 16.8 1.92 RB- B 490 cm² NV 8rpm 45.5 41.5 11.6 1.36 RB- C 490 cm² V 5 rpm 43.6 35.0 20.1 1.34 RB- D490 cm² NV 5 rpm 43.8 42.9 9.71 3.65 Average, RB A-D 43.25 40.1 14.552.07 *V, vented; NV, not vented;

It will be appreciated that the methods and compositions described aboverelate to cells cultured in vitro. However, the above-described in vitrodifferentiated cell compositions may be used for in vivo applications.Use of the compositions described herein have been described detail inat least Applicant's U.S. Pat. Nos. 7,534,608; 7,695,965; and 7,993,920;entitled METHODS FOR PRODUCING PANCREATIC HORMONES, which issued May 19,2009, Apr. 13, 2010 and Aug. 9, 2011, respectively; and 8,278,106,entitled ENCAPSULATION OF PANCREATIC CELLS DERIVED FROM PLURIPOTENT STEMCELLS, the disclosures of which are incorporated herein by reference intheir entireties. Use and function of the compositions described hereinhave also been reported by Applicant in prior non-patent publicationsincluding Kroon et al. 2008 supra and Schulz et al. 2012, supra, whichare also incorporated herein by reference in their entireties.

Accordingly, it will be apparent to one skilled in the art that varyingsubstitutions, modifications or optimization, or combinations may bemade to the embodiments disclosed herein without departing from thescope and spirit of the invention. As described above, roller bottlescan be of varying size, shape and potentially even those containers notcylindrical in shape but which methods simulate the same motion as thatof roller bottles can be used. Further, it is clear from the abovedescription that use of different types of pPSC media, such as XF HA orStemPro® hESC SFM media and other types of media are wholly anticipated,e.g. mTeSR™ media, Essential™ 8 or any other pPSC media commonlyemployed in the industry for growth and culture of pPSC or like cells.

For example, whether aggregation and/or differentiation requires gassing(e.g. CO₂ and other gases) may in part depend on the type of rollerbottle caps used (e.g. vented or not vented). CO₂ can be easilyincorporated into the culture conditions in standard tissue cultureincubators. However, aggregation and/or differentiation with no externalCO₂ (ungassed) can bring certain advantages to manufacturing in scaling:more volume/bottle and therefore fewer bottles per large manufacturingrun. Also, large arrays of bottles could be run in a walk-in hot room ascompared to an incubator for example, greatly simplifying incorporationof robotics and automation in the scaling process.

Certain culture vessels have been described herein (e.g. 6 well trays,bioreactors, Erlenmeyer flasks, roller bottles and the like), howeverother similar culture vessels, for example, those with similar size,shape, dimension and function are contemplated. Commercial rollerbottles, such as Corning's plastic and glass roller bottles, range insizes: 490 cm² (hold 100 to 150 mL); 850 cm² (hold 170 to 255 mL); 1700cm² (hold 340 to 510 mL); 1750 cm² (hold 350 to 525 mL); 670-680 cm²(hold 135 to 200 mL); 840 cm² (hold 170 to 255 mL); 1170 cm² (hold 235to 350 mL); 1330 cm² (hold 265 to 400 mL); 1585 cm² (315 to 475 mL); and1585 cm² (hold 315 to 475 mL).

Erlenmeyer flasks are conical shaped and have a tapered body and narrowneck. The shape, which is distinguished from a beaker, allows thecontents to be swirled or stirred, with an external mechanical device orby hand, while the narrow neck keeps the contents from spilling out andreduces evaporative losses as compared to a beaker, while the flatbottom of the conical flask makes it stable. The invention describedherein contemplates other containers that have similar shapes.

Still the invention described herein also contemplates use ofbioreactors, or any manufactured or engineered device or system thatsupports a biologically active environment. Such bioreactors arecommonly cylindrical, ranging in size from litres to cubic metres. Thereare many commercially available bioreactors and one skilled in the artcan be guided to select the right vessel for their process given thedetailed description provided herein.

Hence, the skilled artisan can easily choose the appropriate size rollerbottle for their scale-up culture needs based on the present inventiondescription and manufacture recommendations.

All publications and patents mentioned in this specification are hereinincorporated in their entireties by reference.

As used in the claims below and throughout this disclosure, by thephrase “consisting essentially of” is meant including any elementslisted after the phrase, and limited to other elements that do notinterfere with or contribute to the activity or action specified in thedisclosure for the listed elements. Thus, the phrase “consistingessentially of” indicates that the listed elements are required ormandatory, but that other elements are optional and may or may not bepresent depending upon whether or not they affect the activity or actionof the listed elements.

What is claimed is:
 1. A roller bottle comprising a culture of primatepluripotent stem cell (pPSC) aggregates suspended in a physiologicallyacceptable medium.
 2. The roller bottle of claim 1, wherein the pPSCaggregates have a diameter of about 100 to 300 microns
 3. The rollerbottle of claim 1, wherein the culture is substantially free of pPSCagglomerations having a diameter of more than 300 microns.
 4. The rollerbottle of claim 1, wherein the pPSC aggregates express at least onemarker selected from the group consisting of OCT4, NANOG, SSEA-3,SSEA-4, Tra-1-80 and Tra-1-60.
 5. The roller bottle of claim 1, whereinthe pPSC aggregates are human cells.
 6. The roller bottle of claim 5,wherein the pPSC aggregates are selected from the group consisting ofhuman embryonic stem cells (hESC) and induced pluripotent stem cells(iPSC).
 7. The roller bottle of claim 1, wherein the primate pluripotentstem cell-derived cell aggregates in suspension are generated by amethod comprising: a. providing a culture of undifferentiated pPSCs; b.seeding a roller bottle with the undifferentiated pPSCs in a culturemedium that supports undifferentiated growth of the pPSCs; and c.agitating the pPSCs at low rotation speed to form pPSC aggregates,thereby generating a roller bottle comprising a culture of pPSCaggregates.
 8. The roller bottle of claim 7, wherein the pPSC aggregateshave a diameter of about 100 to 300 microns.
 9. The roller bottle ofclaim 7, wherein the culture is substantially free of pPSCagglomerations having a diameter of more than 300 microns.
 10. Theroller bottle of claim 7, wherein the pPSC aggregates express at leastone marker selected from the group consisting of OCT4, NANOG, SSEA-3,SSEA-4, Tra-1-80 and Tra-1-60.
 11. The roller bottle of claim 7, whereinthe pPSC aggregates are human cells.
 12. The roller bottle of claim 11,wherein the pPSC aggregates are cells selected from the group consistingof human embryonic stem cells (hESC) and induced pluripotent stem cells(iPSC).
 13. The roller bottle of claim 7, wherein the rotation speedranges from 3 to 20 rpm.
 14. The roller bottle of claim 7, wherein therotation speed ranges from 5 to 12 rpm.
 15. The roller bottle of claim7, wherein the culture of undifferentiated pPSCs in step a is asuspension of single cell pPSCs.
 16. A method for preparing the rollerbottle of claim 1, comprising: a. providing a culture ofundifferentiated pPSCs; b. seeding a roller bottle with theundifferentiated pPSCs in a culture medium that supportsundifferentiated growth of the pPSCs; and c. agitating the single cellsuspension at low rotation speed in a roller bottle to form pPSC cellaggregates, thereby preparing a roller bottle comprising a culture ofpPSC aggregates suspended in a physiologically acceptable medium. 17.The method of claim 16, wherein the culture of undifferentiated pPSCs isa single cell suspension of pPSCs.
 18. The method of claim 16, whereinthe culture of undifferentiated pPSCs is a suspension of thawed pPSCssuspended in a culture medium.
 19. The method of claim 16, wherein therotation speed is about 3 rpm to about 20 rpm.
 20. The method of claim16, wherein the rotation is about 5 rpm to about 20 rpm.
 21. The methodof claim 16, wherein the rotation speed is about 5 rpm to about 12 rpm.22. The method of claim 16, wherein the pPSC is a human cell.
 23. Themethod of claim 22, wherein the pPSC is selected from the groupconsisting of hESC and iPSC.
 24. A method of differentiating primatepluripotent stem cells (pPSCs) in a roller bottle comprising: a.providing a culture of undifferentiated pPSCs; b. agitating the pPSCs ina physiologically acceptable medium in a roller bottle at low rotationspeed to form pPSC aggregates; and c. contacting the pPSC aggregates inthe roller bottle with a culture medium that differentiates the pPSCs toform differentiated cell aggregates, thereby differentiating pPSCs in aroller bottle.
 25. The method of claim 24, wherein the pPSCs aredifferentiated in a feeder-, matrix- and adherent growth culture-freeenvironment.
 26. The method of claim 24, wherein the rotation speed isabout 3 rpm to about 20 rpm.
 27. The method of claim 24, wherein therotation speed is about 5 rpm to about 20 rpm.
 28. The method of claim24 wherein the rotation speed is about 5 rpm to about 12 rpm.
 29. Themethod of claim 24, wherein the pPSCs are human cells.
 30. The method ofclaim 29, wherein the pPSCs are selected from the group consisting ofhESCs and iPSCs.